Maternal educational attainment in pregnancy and epigenome-wide DNA methylation changes in the offspring from birth until adolescence.

Maternal educational attainment (MEA) shapes offspring health through multiple potential pathways. Differential DNA methylation may provide a mechanistic understanding of these long-term associations. We aimed to quantify the associations of MEA with offspring DNA methylation levels at birth, in childhood and in adolescence. Using 37 studies from high-income countries, we performed meta-analysis of epigenome-wide association studies (EWAS) to quantify the associations of completed years of MEA at the time of pregnancy with offspring DNA methylation levels at birth (n = 9 881), in childhood (n = 2 017), and adolescence (n = 2 740), adjusting for relevant covariates. MEA was found to be associated with DNA methylation at 473 cytosine-phosphate-guanine sites at birth, one in childhood, and four in adolescence. We observed enrichment for findings from previous EWAS on maternal folate, vitamin-B12 concentrations, maternal smoking, and pre-pregnancy BMI. The associations were directionally consistent with MEA being inversely associated with behaviours including smoking and BMI. Our findings form a bridge between socio-economic factors and biology and highlight potential pathways underlying effects of maternal education. The results broaden our understanding of bio-social associations linked to differential DNA methylation in multiple early stages of life. The data generated also offers an important resource to help a more precise understanding of the social determinants of health.

Adam19 Deficiency Impacts Pulmonary Function: Human GWAS Follow-up in a Mouse Knockout Model.

PURPOSE: Over 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function. METHODS: We created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent. RESULTS: Contrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways. CONCLUSION: Our murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.

High-throughput screening and genome-wide analyses of 44 anticancer drugs in the 1000 Genomes cell lines reveals an association of the NQO1 gene with the response of multiple anticancer drugs.

Cancer patients exhibit a broad range of inter-individual variability in response and toxicity to widely used anticancer drugs, and genetic variation is a major contributor to this variability. To identify new genes that influence the response of 44 FDA-approved anticancer drug treatments widely used to treat various types of cancer, we conducted high-throughput screening and genome-wide association mapping using 680 lymphoblastoid cell lines from the 1000 Genomes Project. The drug treatments considered in this study represent nine drug classes widely used in the treatment of cancer in addition to the paclitaxel + epirubicin combination therapy commonly used for breast cancer patients. Our genome-wide association study (GWAS) found several significant and suggestive associations. We prioritized consistent associations for functional follow-up using gene-expression analyses. The NAD(P)H quinone dehydrogenase 1 (NQO1) gene was found to be associated with the dose-response of arsenic trioxide, erlotinib, trametinib, and a combination treatment of paclitaxel + epirubicin. NQO1 has previously been shown as a biomarker of epirubicin response, but our results reveal novel associations with these additional treatments. Baseline gene expression of NQO1 was positively correlated with response for 43 of the 44 treatments surveyed. By interrogating the functional mechanisms of this association, the results demonstrate differences in both baseline and drug-exposed induction.

Multi-tissue transcriptomic and serum metabolomic assessment reveals systemic implications of acute ozone-induced stress response in male Wistar Kyoto rats.

Air pollutant exposures have been linked to systemic disease; however, the underlying mechanisms between responses of the target tissue and systemic effects are poorly understood. A prototypic inducer of stress, ozone causes respiratory and systemic multiorgan effects through activation of a neuroendocrine stress response. The goal of this study was to assess transcriptomic signatures of multiple tissues and serum metabolomics to understand how neuroendocrine and adrenal-derived stress hormones contribute to multiorgan health outcomes. Male Wistar Kyoto rats (12-13 weeks old) were exposed to filtered air or 0.8 ppm ozone for 4-hours, and blood/tissues were collected immediately post-exposure. Each tissue had distinct expression profiles at baseline. Ozone changed 1,640 genes in lung, 274 in hypothalamus, 2,516 in adrenals, 1,333 in liver, 1,242 in adipose, and 5,102 in muscle (adjusted p-value < 0.1, absolute fold-change > 50%). Serum metabolomic analysis identified 863 metabolites, of which 447 were significantly altered in ozone-exposed rats (adjusted p-value < 0.1, absolute fold change > 20%). A total of 6 genes were differentially expressed in all 6 tissues. Glucocorticoid signaling, hypoxia, and GPCR signaling were commonly changed, but ozone induced tissue-specific changes in oxidative stress, immune processes, and metabolic pathways. Genes upregulated by TNF-mediated NFkB signaling were differentially expressed in all ozone-exposed tissues, but those defining inflammatory response were tissue-specific. Upstream predictor analysis identified common mediators of effects including glucocorticoids, although the specific genes responsible for these predictors varied by tissue. Metabolomic analysis showed major changes in lipids, amino acids, and metabolites linked to the gut microbiome, concordant with transcriptional changes identified through pathway analysis within liver, muscle, and adipose tissues. The distribution of receptors and transcriptional mechanisms underlying the ozone-induced stress response are tissue-specific and involve induction of unique gene networks and metabolic phenotypes, but the shared initiating triggers converge into shared pathway-level responses. This multi-tissue transcriptomic analysis, combined with circulating metabolomic assessment, allows characterization of the systemic inhaled pollutant-induced stress response.

Social isolation exacerbates acute ozone inhalation induced pulmonary and systemic health outcomes.

Psychosocially-stressed individuals might have exacerbated responses to air pollution exposure. Acute ozone exposure activates the neuroendocrine stress response leading to systemic metabolic and lung inflammatory changes. We hypothesized chronic mild stress (CS) and/or social isolation (SI) would cause neuroendocrine, inflammatory, and metabolic phenotypes that would be exacerbated by an acute ozone exposure. Male 5-week-old Wistar-Kyoto rats were randomly assigned into 3 groups: no stress (NS) (pair-housed, regular-handling); SI (single-housed, minimal-handling); CS (single-housed, subjected to mild unpredicted-randomized stressors [restraint-1 h, tilted cage-1 h, shaking-1 h, intermittent noise-6 h, and predator odor-1 h], 1-stressor/day*5-days/week*8-weeks. All animals then 13-week-old were subsequently exposed to filtered-air or ozone (0.8-ppm) for 4 h and immediately necropsied. CS, but not SI animals had increased adrenal weights. However, relative to NS, both CS and SI had lower circulating luteinizing hormone, prolactin, and follicle-stimulating hormone regardless of exposure (SI > CS), and only CS demonstrated lower thyroid-stimulating hormone levels. SI caused more severe systemic inflammation than CS, as evidenced by higher circulating cytokines and cholesterol. Ozone exposure increased urine corticosterone and catecholamine metabolites with no significant stressor effect. Ozone-induced lung injury, and increases in lavage-fluid neutrophils and IL-6, were exacerbated by SI. Ozone severely lowered circulating thyroid-stimulating hormone, prolactin, and luteinizing hormone in all groups and exacerbated systemic inflammation in SI. Ozone-induced increases in serum glucose, leptin, and triglycerides were consistent across stressors; however, increases in cholesterol were exacerbated by SI. Collectively, psychosocial stressors, especially SI, affected the neuroendocrine system and induced adverse metabolic and inflammatory effects that were exacerbated by ozone exposure.

Questionnaire-based exposome-wide association studies (ExWAS) reveal expected and novel risk factors associated with cardiovascular outcomes in the Personalized Environment and Genes Study.

While multiple factors are associated with cardiovascular disease (CVD), many environmental exposures that may contribute to CVD have not been examined. To understand environmental effects on cardiovascular health, we performed an exposome-wide association study (ExWAS), a hypothesis-free approach, using survey data on endogenous and exogenous exposures at home and work and data from health and medical histories from the North Carolina-based Personalized Environment and Genes Study (PEGS) (n = 5015). We performed ExWAS analyses separately on six cardiovascular outcomes (cardiac arrhythmia, congestive heart failure, coronary artery disease, heart attack, stroke, and a combined atherogenic-related outcome comprising angina, angioplasty, atherosclerosis, coronary artery disease, heart attack, and stroke) using logistic regression and a false discovery rate of 5%. For each CVD outcome, we tested 502 single exposures and built multi-exposure models using the deletion-substitution-addition (DSA) algorithm. To evaluate complex nonlinear relationships, we employed the knockoff boosted tree (KOBT) algorithm. We adjusted all analyses for age, sex, race, BMI, and annual household income. ExWAS analyses revealed novel associations that include blood type A (Rh-) with heart attack (OR[95%CI] = 8.2[2.2:29.7]); paint exposures with stroke (paint related chemicals: 6.1[2.2:16.0], acrylic paint: 8.1[2.6:22.9], primer: 6.7[2.2:18.6]); biohazardous materials exposure with arrhythmia (1.8[1.5:2.3]); and higher paternal education level with reduced risk of multiple CVD outcomes (stroke, heart attack, coronary artery disease, and combined atherogenic outcome). In multi-exposure models, trouble sleeping and smoking remained important risk factors. KOBT identified significant nonlinear effects of sleep disorder, regular intake of grapefruit, and a family history of blood clotting problems for multiple CVD outcomes (combined atherogenic outcome, congestive heart failure, and coronary artery disease). In conclusion, using statistics and machine learning, these findings identify novel potential risk factors for CVD, enable hypothesis generation, provide insights into the complex relationships between risk factors and CVD, and highlight the importance of considering multiple exposures when examining CVD outcomes.

Clomifene and Assisted Reproductive Technology in Humans Are Associated with Sex-Specific Offspring Epigenetic Alterations in Imprinted Control Regions.

Children conceived with assisted reproductive technology (ART) have an increased risk of adverse outcomes, including congenital malformations and imprinted gene disorders. In a retrospective North Carolina-based-birth-cohort, we examined the effect of ovulation drugs and ART on CpG methylation in differentially methylated CpGs in known imprint control regions (ICRs). Nine ICRs containing 48 CpGs were assessed for methylation status by pyrosequencing in mixed leukocytes from cord blood. After restricting to non-smoking, college-educated participants who agreed to follow-up, ART-exposed (n = 27), clomifene-only-exposed (n = 22), and non-exposed (n = 516) groups were defined. Associations of clomifene and ART with ICR CpG methylation were assessed with linear regression and stratifying by offspring sex. In males, ART was associated with hypomethylation of the PEG3 ICR [ß(95% CI) = -1.46 (-2.81, -0.12)] and hypermethylation of the MEG3 ICR [3.71 (0.01, 7.40)]; clomifene-only was associated with hypomethylation of the NNAT ICR [-5.25 (-10.12, -0.38)]. In female offspring, ART was associated with hypomethylation of the IGF2 ICR [-3.67 (-6.79, -0.55)]. Aberrant methylation of these ICRs has been associated with cardiovascular disease and metabolic and behavioral outcomes in children. The results suggest that the increased risk of adverse outcomes in offspring conceived through ART may be due in part to altered methylation of ICRs. Larger studies utilizing epigenome-wide interrogation are warranted.

Lrp1 Regulation of Pulmonary Function. Follow-Up of Human GWAS in Mice.

Human genome-wide association studies (GWASs) have identified more than 270 loci associated with pulmonary function; however, follow-up studies to determine causal genes at these loci are few. SNPs in low-density lipoprotein receptor-related protein 1 (LRP1) are associated with human pulmonary function in GWASs. Using murine models, we investigated the effect of genetic disruption of the Lrp1 gene in smooth muscle cells on pulmonary function in naive animals and after exposure to bacterial LPS or house dust mite extract. Disruption of Lrp1 in smooth muscle cells leads to an increase in tissue resistance, elastance, and tissue elastance at baseline. Furthermore, disruption of Lrp1 in smooth muscle increases airway responsiveness as measured by increased total lung resistance and airway resistance after methacholine. Immune cell counts in BAL fluid were increased in animals with Lrp1 disruption. The difference in airway responsiveness by genotype observed in naive animals was not observed after LPS or house dust mite extract exposure. To further explore the mechanisms contributing to changes in pulmonary function, we identified several ligands dysregulated with Lrp1 disruption in smooth muscle cells. These data suggest that dysregulation of LRP1 in smooth muscle cells affects baseline pulmonary function and airway responsiveness and helps establish LRP1 as the causal gene at this GWAS locus.

Peripheral metabolic effects of ozone exposure in healthy and diabetic rats on normal or high-cholesterol diet.

Epidemiological studies show that individuals with underlying diabetes and diet-associated ailments are more susceptible than healthy individuals to adverse health effects of air pollution. Exposure to air pollutants can induce metabolic stress and increase cardiometabolic disease risk. Using male Wistar and Wistar-derived Goto-Kakizaki (GK) rats, which exhibit a non-obese type-2 diabetes phenotype, we investigated whether two key metabolic stressors, type-2 diabetes and a high-cholesterol atherogenic diet, exacerbate ozone-induced metabolic effects. Rats were fed a normal control diet (ND) or high-cholesterol diet (HCD) for 12 weeks and then exposed to filtered air or 1.0-ppm ozone (6 h/day) for 1 or 2 days. Metabolic responses were analyzed at the end of each day and after an 18-h recovery period following the 2-day exposure. In GK rats, baseline hyperglycemia and glucose intolerance were exacerbated by HCD vs. ND and by ozone vs. air. HCD also resulted in higher insulin in ozone-exposed GK rats and circulating lipase, aspartate transaminase, and alanine transaminase in all groups (Wistar>GK). Histopathological effects induced by HCD in the liver, which included macrovesicular vacuolation and hepatocellular necrosis, were more severe in Wistar vs. GK rats. Liver gene expression in Wistar and GK rats fed ND showed numerous strain differences, including evidence of increased lipid metabolizing activity and ozone-induced alterations in glucose and lipid transporters, specifically in GK rats. Collectively, these findings indicate that peripheral metabolic alterations induced by diabetes and high-cholesterol diet can enhance susceptibility to the metabolic effects of inhaled pollutants.

Low-Dose Silver Nanoparticle Surface Chemistry and Temporal Effects on Gene Expression in Human Liver Cells.

Silver nanoparticles (AgNPs) are widely incorporated into consumer and biomedical products for their antimicrobial and plasmonic properties with limited risk assessment of low-dose cumulative exposure in humans. To evaluate cellular responses to low-dose AgNP exposures across time, human liver cells (HepG2) are exposed to AgNPs with three different surface charges (1.2 µg mL-1 ) and complete gene expression is monitored across a 24 h period. Time and AgNP surface chemistry mediate gene expression. In addition, since cells are fed, time has marked effects on gene expression that should be considered. Surface chemistry of AgNPs alters gene transcription in a time-dependent manner, with the most dramatic effects in cationic AgNPs. Universal to all surface coatings, AgNP-treated cells responded by inactivating proliferation and enabling cell cycle checkpoints. Further analysis of these universal features of AgNP cellular response, as well as more detailed analysis of specific AgNP treatments, time points, or specific genes, is facilitated with an accompanying application. Taken together, these results provide a foundation for understanding hepatic response to low-dose AgNPs for future risk assessment.

C/EBPß suppresses keratinocyte autonomous type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer.

p53 is activated by DNA damage and oncogenic stimuli to regulate senescence, apoptosis and cell-cycle arrest, which are essential to prevent cancer. Here, we utilized UVB radiation, a potent inducer of DNA damage, p53, apoptosis and skin cancer to investigate the mechanism of CCAAT/enhancer binding protein-ß (C/EBPß) in regulating p53-mediated apoptosis in keratinocytes and to test whether the deletion of C/EBPß in epidermis can protect mice from UVB-induced skin cancer. UVB-treatment of C/EBPß skin conditional knockout (CKOß) mice increased p53 protein levels in epidermis and enhanced p53-dependent apoptotic activity 3-fold compared with UVB-treated control mice. UVB increased C/EBPß levels through a p53-dependent pathway and stimulated the formation of a C/EBPß-p53 protein complex; knockdown of C/EBPß increased p53 protein stability in keratinocytes. These results suggest a p53-C/EBPß feedback loop, whereby C/EBPß, a transcriptional target of a p53 pathway, functions as a survival factor by negatively regulating p53 apoptotic activity in response to DNA damage. RNAseq analysis of UVB-treated CKOß epidermis unexpectedly revealed that type 1 interferon (IFN) pathway was the most highly enriched pathway. Numerous pro-apoptotic interferon stimulated genes were upregulated including some known to enhance p53 apoptosis. Our results indicate that p53 and IFN pathways function together in response to DNA damage to result in the activation of extrinsic apoptosis pathways and caspase 8 cleavage. Last, we observed CKOß mice were resistant to UVB-induced skin cancer. Our results suggest that C/EBPß represses apoptosis through keratinocyte autonomous suppression of the type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer.

Population-based toxicity screening in human induced pluripotent stem cell-derived cardiomyocytes.

The potential for cardiotoxicity is carefully evaluated for pharmaceuticals, as it is a major safety liability. However, environmental chemicals are seldom tested for their cardiotoxic potential. Moreover, there is a large variability in both baseline and drug-induced cardiovascular risk in humans, but data are lacking on the degree to which susceptibility to chemically-induced cardiotoxicity may also vary. Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes have become an important in vitro model for drug screening. Thus, we hypothesized that a population-based model of iPSC-derived cardiomyocytes from a diverse set of individuals can be used to assess potential hazard and inter-individual variability in chemical effects on these cells. We conducted concentration-response screening of 134 chemicals (pharmaceuticals, industrial and environmental chemicals and food constituents) in iPSC-derived cardiomyocytes from 43 individuals, comprising both sexes and diverse ancestry. We measured kinetic calcium flux and conducted high-content imaging following chemical exposure, and utilized a panel of functional and cytotoxicity parameters in concentration-response for each chemical and donor. We show reproducible inter-individual variability in both baseline and chemical-induced effects on iPSC-derived cardiomyocytes. Further, chemical-specific variability in potency and degree of population variability were quantified. This study shows the feasibility of using an organotypic population-based human in vitro model to quantitatively assess chemicals for which little cardiotoxicity information is available. Ultimately, these results advance in vitro toxicity testing methodologies by providing an innovative tool for population-based cardiotoxicity screening, contributing to the paradigm shift from traditional animal models of toxicity to in vitro toxicity testing methods.

Multi-dimensional in vitro bioactivity profiling for grouping of glycol ethers.

High-content screening data derived from physiologically-relevant in vitro models promise to improve confidence in data-integrative groupings for read-across in human health safety assessments. The biological data-based read-across concept is especially applicable to bioactive chemicals with defined mechanisms of toxicity; however, the challenge of data-derived groupings for chemicals that are associated with little or no bioactivity has not been explored. In this study, we apply a suite of organotypic and population-based in vitro models for comprehensive bioactivity profiling of twenty E-Series and P-Series glycol ethers, solvents with a broad variation in toxicity ranging from relatively non-toxic to reproductive and hematopoetic system toxicants. Both E-Series and P-Series glycol ethers elicited cytotoxicity only at high concentrations (mM range) in induced pluripotent stem cell-derived hepatocytes and cardiomyocytes. Population-variability assessment comprised a study of cytotoxicity in 94 human lymphoblast cell lines from 9 populations and revealed differences in inter-individual variability across glycol ethers, but did not indicate population-specific effects. Data derived from various phenotypic and transcriptomic assays revealed consistent bioactivity trends between both cardiomyocytes and hepatocytes, indicating a more universal, rather than cell-type specific mode-of-action for the tested glycol ethers in vitro. In vitro bioactivity-based similarity assessment using Toxicological Priority Index (ToxPi) showed that glycol ethers group according to their alcohol chain length, longer chains were associated with increased bioactivity. While overall in vitro bioactivity profiles did not correlate with in vivo toxicity data on glycol ethers, in vitro bioactivity of E-series glycol ethers were indicative of and correlated with in vivo irritation scores.

Raw milk consumption and other early-life farm exposures and adult pulmonary function in the Agricultural Lung Health Study.

Literature suggests that early exposure to the farming environment protects against atopy and asthma; few studies have examined pulmonary function. We evaluated associations between early-life farming exposures and pulmonary function in 3061 adults (mean age=63) from a US farming population using linear regression. Childhood raw milk consumption was associated with higher FEV1 (ß=49.5 mL, 95% CI 2.8 to 96.1 mL, p=0.04) and FVC (ß=66.2 mL, 95% CI 13.2 to 119.1 mL, p=0.01). We did not find appreciable associations with other early-life farming exposures. We report a novel association between raw milk consumption and higher pulmonary function that lasts into older adulthood.

Population-based dose-response analysis of liver transcriptional response to trichloroethylene in mouse.

Studies of gene expression are common in toxicology and provide important clues to mechanistic understanding of adverse effects of chemicals. Most prior studies have been performed in a single strain or cell line; however, gene expression is heavily influenced by the genetic background, and these genotype-expression differences may be key drivers of inter-individual variation in response to chemical toxicity. In this study, we hypothesized that the genetically diverse Collaborative Cross mouse population can be used to gain insight and suggest mechanistic hypotheses for the dose- and genetic background-dependent effects of chemical exposure. This hypothesis was tested using a model liver toxicant trichloroethylene (TCE). Liver transcriptional responses to TCE exposure were evaluated 24 h after dosing. Transcriptomic dose-responses were examined for both TCE and its major oxidative metabolite trichloroacetic acid (TCA). As expected, peroxisome- and fatty acid metabolism-related pathways were among the most dose-responsive enriched pathways in all strains. However, nearly half of the TCE-induced liver transcriptional perturbation was strain-dependent, with abundant evidence of strain/dose interaction, including in the peroxisomal signaling-associated pathways. These effects were highly concordant between the administered TCE dose and liver levels of TCA. Dose-response analysis of gene expression at the pathway level yielded points of departure similar to those derived from the traditional toxicology studies for both non-cancer and cancer effects. Mapping of expression-genotype-dose relationships revealed some significant associations; however, the effects of TCE on gene expression in liver appear to be highly polygenic traits that are challenging to positionally map. This study highlights the usefulness of mouse population-based studies in assessing inter-individual variation in toxicological responses, but cautions that genetic mapping may be challenging because of the complexity in gene exposure-dose relationships.

Early-life farm exposures and adult asthma and atopy in the Agricultural Lung Health Study.

BACKGROUND: Previous studies, mostly from Europe, suggest that early-life farming exposures protect against childhood asthma and allergy; few data exist on asthma and allergy in adults. OBJECTIVE: We sought to examine associations between early-life farming exposures and current asthma and atopy in an older adult US farming population. METHODS: We analyzed data from 1746 farmers and 1555 spouses (mean age, 63) from a case-control study nested within the Agricultural Health Study. Current asthma and early-life farming exposures were assessed via questionnaires. We defined atopy based on specific IgE > 0.70 IU/mL to at least 1 of 10 allergens measured in blood. We used logistic regression, adjusted for age, sex, race, state (Iowa or North Carolina), and smoking (pack years), to estimate associations between early-life exposures and asthma (1198 cases and 2031 noncases) or atopy (578 cases and 2526 noncases). RESULTS: Exposure to the farming environment in utero and in early childhood had little or no association with asthma but was associated with reduced odds of atopy. The strongest association was seen for having a mother who performed farm activities while pregnant (odds ratio, 0.60; 95% CI, 0.48-0.74) and remained significant in models with correlated early-life exposures including early childhood farm animal contact and raw milk consumption. CONCLUSIONS: In a large US farming population, early-life farm exposures, particularly maternal farming activities while pregnant, were strongly associated with reduced risk of atopy in adults. These results extend previous work done primarily on childhood outcomes and suggest that protective associations of early-life farming exposures on atopy endure across the life course.

Vagal innervation is required for pulmonary function phenotype in Htr4-/- mice.

Human genome-wide association studies have identified over 50 loci associated with pulmonary function and related phenotypes, yet follow-up studies to determine causal genes or variants are rare. Single nucleotide polymorphisms in serotonin receptor 4 (HTR4) are associated with human pulmonary function in genome-wide association studies and follow-up animal work has demonstrated that Htr4 is causally associated with pulmonary function in mice, although the precise mechanisms were not identified. We sought to elucidate the role of neural innervation and pulmonary architecture in the lung phenotype of Htr4-/- animals. We report here that the Htr4-/- phenotype in mouse is dependent on vagal innervation to the lung. Both ex vivo tracheal ring reactivity and in vivo flexiVent pulmonary functional analyses demonstrate that vagotomy abrogates the Htr4-/- airway hyperresponsiveness phenotype. Hyperpolarized 3He gas magnetic resonance imaging and stereological assessment of wild-type and Htr4-/- mice reveal no observable differences in lung volume, inflation characteristics, or pulmonary microarchitecture. Finally, control of breathing experiments reveal substantive differences in baseline breathing characteristics between mice with/without functional HTR4 in breathing frequency, relaxation time, flow rate, minute volume, time of inspiration and expiration and breathing pauses. These results suggest that HTR4's role in pulmonary function likely relates to neural innervation and control of breathing.

Genetic variation in HTR4 and lung function: GWAS follow-up in mouse.

Human genome-wide association studies (GWASs) have identified numerous associations between single nucleotide polymorphisms (SNPs) and pulmonary function. Proving that there is a causal relationship between GWAS SNPs, many of which are noncoding and without known functional impact, and these traits has been elusive. Furthermore, noncoding GWAS-identified SNPs may exert trans-regulatory effects rather than impact the proximal gene. Noncoding variants in 5-hydroxytryptamine (serotonin) receptor 4 (HTR4) are associated with pulmonary function in human GWASs. To gain insight into whether this association is causal, we tested whether Htr4-null mice have altered pulmonary function. We found that HTR4-deficient mice have 12% higher baseline lung resistance and also increased methacholine-induced airway hyperresponsiveness (AHR) as measured by lung resistance (27%), tissue resistance (48%), and tissue elastance (30%). Furthermore, Htr4-null mice were more sensitive to serotonin-induced AHR. In models of exposure to bacterial lipopolysaccharide, bleomycin, and allergic airway inflammation induced by house dust mites, pulmonary function and cytokine profiles in Htr4-null mice differed little from their wild-type controls. The findings of altered baseline lung function and increased AHR in Htr4-null mice support a causal relationship between genetic variation in HTR4 and pulmonary function identified in human GWAS.

Quantitative Polymerase Chain Reaction Analysis of the Mouse Cyp2j Subfamily: Tissue Distribution and Regulation.

Members of the cytochrome P450 CYP2J subfamily are expressed in multiple tissues in mice and humans. These enzymes are active in the metabolism of fatty acids to generate bioactive compounds. Herein we report new methods and results for quantitative polymerase chain reaction (qPCR) analysis for the seven genes (Cyp2j5, Cyp2j6, Cyp2j8, Cyp2j9, Cyp2j11, Cyp2j12, and Cyp2j13) of the mouse Cyp2j subfamily. SYBR Green primer sets were developed and compared with commercially available TaqMan primer/probe assays for specificity toward mouse Cyp2j cDNA, and analysis of tissue distribution and regulation of Cyp2j genes. Each TaqMan primer/probe set and SYBR Green primer set were shown to be specific for their intended mouse Cyp2j cDNA. Tissue distribution of the mouse Cyp2j isoforms confirmed similar patterns of expression between the two qPCR methods. Cyp2j5 and Cyp2j13 were highly expressed in male kidneys, and Cyp2j11 was highly expressed in both male and female kidneys. Cyp2j6 was expressed in multiple tissues, with the highest expression in the small intestine and duodenum. Cyp2j8 was detected in various tissues, with highest expression found in the skin. Cyp2j9 was highly expressed in the brain, liver, and lung. Cyp2j12 was predominately expressed in the brain. We also determined the Cyp2j isoform expression in Cyp2j5 knockout mice to determine whether there was compensatory regulation of other Cyp2j isoforms, and we assessed Cyp2j isoform regulation during various inflammatory models, including influenza A, bacterial lipopolysaccharide, house dust mite allergen, and corn pollen. Both qPCR methods detected similar suppression of Cyp2j6 and Cyp2j9 during inflammation in the lung.

Guided optimization of ToxPi model weights using a Semi-Automated approach.

The Toxicological Prioritization Index (ToxPi) is a visual analysis and decision support tool for dimension reduction and visualization of high throughput, multi-dimensional feature data. ToxPi was originally developed for assessing the relative toxicity of multiple chemicals or stressors by synthesizing complex toxicological data to provide a single comprehensive view of the potential health effects. It continues to be used for profiling chemicals and has since been applied to other types of "sample" entities, including geospatial (e.g. county-level Covid-19 risk and sites of historical PFAS exposure) and other profiling applications. For any set of features (data collected on a set of sample entities), ToxPi integrates the data into a set of weighted slices that provide a visual profile and a score metric for comparison. This scoring system is highly dependent on user-provided feature weights, yet users often lack knowledge of how to define these feature weights. Common methods for predicting feature weights are generally unusable due to inappropriate statistical assumptions and lack of global distributional expectation. However, users often have an inherent understanding of expected results for a small subset of samples. For example, in chemical toxicity, prior knowledge can often place subsets of chemicals into categories of low, moderate or high toxicity (reference chemicals). Ordinal regression can be used to predict weights based on these response levels that are applicable to the entire feature set, analogous to using positive and negative controls to contextualize an empirical distribution. We propose a semi-supervised method utilizing ordinal regression to predict a set of feature weights that produces the best fit for the known response ("reference") data and subsequently fine-tunes the weights via a customized genetic algorithm. We conduct a simulation study to show when this method can improve the results of ordinal regression, allowing for accurate feature weight prediction and sample ranking in scenarios with minimal response data. To ground-truth the guided weight optimization, we test this method on published data to build a ToxPi model for comparison against expert-knowledge-driven weight assignments.