Integrating 5hmC and gene expression data to infer regulatory mechanisms.

Motivation: Epigenetic mechanisms are known to play a major role in breast cancer. However, the role of 5-hydroxymethylcytosine (5hmC) remains understudied. We hypothesize that 5hmC mediates redox regulation of gene expression in an aggressive subtype known as triple negative breast cancer (TNBC). To address this, our objective was to highlight genes that may be the target of this process by identifying redox-regulated, antioxidant-sensitive, gene-localized 5hmC changes associated with mRNA changes in TNBC cells. Results: We proceeded to develop an approach to integrate novel Pvu-sequencing and RNA-sequencing data. The result of our approach to merge genome-wide, high-throughput TNBC cell line datasets to identify significant, concordant 5hmC and mRNA changes in response to antioxidant treatment produced a gene set with relevance to cancer stem cell function. Moreover, we have established a method that will be useful for continued research of 5hmC in TNBC cells and tissue samples. Availability and implementation: Data are available at Gene Expression Omnibus (GEO) under accession number GSE103850. Contact: bollig@karmanos.org.

Size-Dependent Toxicity of Gold Nanoparticles on Human Embryonic Stem Cells and Their Neural Derivatives.

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5, 4, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate-capped AuNPs exhibit loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 µg mL(-1). The cells exposed to 1.5 nm AuNPs at this concentration do not form embryoid bodies but rather disintegrate into single cells within 48 h. Cell death caused by 1.5 nm AuNPs also occur in hESC-derived neural progenitor cells. None of the other nanoparticles exhibit toxic effects on the hESCs at concentrations as high as 10 µg mL(-1) during a 19 d neural differentiation period. Thiolate-capped 4 nm AuNPs at 10 µg mL(-1) cause a dramatic decrease in global DNA methylation (5 mC) and a corresponding increase in global DNA hydroxymethylation (5 hmC) of the hESC's DNA in only 24 h. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells.

Translational toxicoepigenetic Meta-Analyses identify homologous gene DNA methylation reprogramming following developmental phthalate and lead exposure in mouse and human offspring.

Although toxicology uses animal models to represent real-world human health scenarios, a critical translational gap between laboratory-based studies and epidemiology remains. In this study, we aimed to understand the toxicoepigenetic effects on DNA methylation after developmental exposure to two common toxicants, the phthalate di(2-ethylhexyl) phthalate (DEHP) and the metal lead (Pb), using a translational paradigm that selected candidate genes from a mouse study and assessed them in four human birth cohorts. Data from mouse offspring developmentally exposed to DEHP, Pb, or control were used to identify genes with sex-specific sites with differential DNA methylation at postnatal day 21. Associations of human infant DNA methylation in homologous mouse genes with prenatal DEHP or Pb were examined with a meta-analysis. Differential methylation was observed on 6 cytosines (adjusted-p < 0.05) and 90 regions (adjusted-p < 0.001). This translational approach offers a unique method that can detect conserved epigenetic differences that are developmentally susceptible to environmental toxicants.

Prenatal Phenol and Paraben Exposures and Adverse Birth Outcomes: A Prospective Analysis of U.S. Births.

BACKGROUND: Synthetic chemicals are increasingly being recognized for potential independent contributions to preterm birth (PTB) and low birth weight (LBW). Bisphenols, parabens, and triclosan are consumer product chemicals that act via similar mechanisms including estrogen, androgen, and thyroid disruption and oxidative stress. Multiple cohort studies have endeavored to examine effects on birth outcomes, and systematic reviews have been limited due to measurement of 1-2 spot samples during pregnancy and limited diversity of populations. OBJECTIVE: To study the effects of prenatal phenols and parabens on birth size and gestational age (GA) in 3,619 mother-infant pairs from 11 cohorts in the NIH Environmental influences on Child Health Outcomes program. RESULTS: While many associations were modest and statistically imprecise, a 1-unit increase in log10 pregnancy averaged concentration of benzophenone-3 and methylparaben were associated with decreases in birthweight, birthweight adjusted for gestational age and SGA. Increases in the odds of being SGA were 29% (95% CI: 5%, 58%) and 32% (95% CI: 3%, 70%), respectively. Bisphenol S in third trimester was also associated with SGA (OR 1.52, 95% CI 1.08, 2.13). Associations of benzophenone-3 and methylparaben with PTB and LBW were null. In addition, a 1-unit increase in log10 pregnancy averaged concentration of 2,4-dichlorophenol was associated with 43% lower (95% CI: -67%, -2%) odds of low birthweight; the direction of effect was the same for the highly correlated 2,5-dichlorophenol, but with a smaller magnitude (-29%, 95% CI: -53%, 8%). DISCUSSION: In a large and diverse sample generally representative of the United States, benzophenone-3 and methylparaben were associated with lower birthweight as well as birthweight adjusted for gestational age and higher odds of SGA, while 2,4-dichlorophenol. These associations with smaller size at birth are concerning in light of the known consequences of intrauterine growth restriction for multiple important health outcomes emerging later in life.

Prenatal phthalate exposure and adverse birth outcomes in the USA: a prospective analysis of births and estimates of attributable burden and costs.

BACKGROUND: Phthalates are synthetic chemicals widely used in consumer products and have been identified to contribute to preterm birth. Existing studies have methodological limitations and potential effects of di-2-ethylhexyl phthalate (DEHP) replacements are poorly characterised. Attributable fractions and costs have not been quantified, limiting the ability to weigh trade-offs involved in ongoing use. We aimed to leverage a large, diverse US cohort to study associations of phthalate metabolites with birthweight and gestational age, and estimate attributable adverse birth outcomes and associated costs. METHODS: In this prospective analysis we used extant data in the US National Institutes of Health Environmental influences on Child Health Outcomes (ECHO) Program from 1998 to 2022 to study associations of 20 phthalate metabolites with gestational age at birth, birthweight, birth length, and birthweight for gestational age z-scores. We also estimated attributable adverse birth outcomes and associated costs. Mother-child dyads were included in the study if there were one or more urinary phthalate measurements during the index pregnancy; data on child's gestational age and birthweight; and singleton delivery. FINDINGS: We identified 5006 mother-child dyads from 13 cohorts in the ECHO Program. Phthalic acid, diisodecyl phthalate (DiDP), di-n-octyl phthalate (DnOP), and diisononyl phthalate (DiNP) were most strongly associated with gestational age, birth length, and birthweight, especially compared with DEHP or other metabolite groupings. Although DEHP was associated with preterm birth (odds ratio 1·45 [95% CI 1·05-2·01]), the risks per log10 increase were higher for phthalic acid (2·71 [1·91-3·83]), DiNP (2·25 [1·67-3·00]), DiDP (1·69 [1·25-2·28]), and DnOP (2·90 [1·96-4·23]). We estimated 56 595 (sensitivity analyses 24 003-120 116) phthalate-attributable preterm birth cases in 2018 with associated costs of US$3·84 billion (sensitivity analysis 1·63- 8·14 billion). INTERPRETATION: In a large, diverse sample of US births, exposure to DEHP, DiDP, DiNP, and DnOP were associated with decreased gestational age and increased risk of preterm birth, suggesting substantial opportunities for prevention. This finding suggests the adverse consequences of substitution of DEHP with chemically similar phthalates and need to regulate chemicals with similar properties as a class. FUNDING: National Institutes of Health.

Prenatal Exposure to Nonpersistent Environmental Chemicals and Postpartum Depression.

Importance: Postpartum depression (PPD) affects up to 20% of childbearing individuals, and a significant limitation in reducing its morbidity is the difficulty in modifying established risk factors. Exposure to synthetic environmental chemicals found in plastics and personal care products, such as phenols, phthalates, and parabens, are potentially modifiable and plausibly linked to PPD and have yet to be explored. Objective: To evaluate associations of prenatal exposure to phenols, phthalates, parabens, and triclocarban with PPD symptoms. Design, Setting, and Participants: This was a prospective cohort study from 5 US sites, conducted from 2006 to 2020, and included pooled data from 5 US birth cohorts from the National Institutes of Health Environmental Influences on Child Health Outcomes (ECHO) consortium. Participants were pregnant individuals with data on urinary chemical concentrations (phenols, phthalate metabolites, parabens, or triclocarban) from at least 1 time point in pregnancy and self-reported postnatal depression screening assessment collected between 2 weeks and 12 months after delivery. Data were analyzed from February to May 2022. Exposures: Phenols (bisphenols and triclosan), phthalate metabolites, parabens, and triclocarban measured in prenatal urine samples. Main Outcomes and Measures: Depression symptom scores were assessed using the Edinburgh Postnatal Depression Scale (EPDS) or the Center for Epidemiologic Studies Depression Scale (CES-D), harmonized to the Patient-Reported Measurement Information System (PROMIS) Depression scale. Measures of dichotomous PPD were created using both sensitive (EPDS scores ≥10 and CES-D scores ≥16) and specific (EPDS scores ≥13 and CES-D scores ≥20) definitions. Results: Among the 2174 pregnant individuals eligible for analysis, nearly all (>99%) had detectable levels of several phthalate metabolites and parabens. PPD was assessed a mean (SD) of 3 (2.5) months after delivery, with 349 individuals (16.1%) and 170 individuals (7.8%) screening positive for PPD using the sensitive and specific definitions, respectively. Linear regression results of continuous PROMIS depression T scores showed no statistically significant associations with any chemical exposures. Models examining LMW and HMW phthalates and di (2-ethylhexyl) phthalate had estimates in the positive direction whereas all others were negative. A 1-unit increase in log-transformed LMW phthalates was associated with a 0.26-unit increase in the PROMIS depression T score (95% CI, -0.01 to 0.53; P = .06). This corresponded to an odds ratio (OR) of 1.08 (95% CI, 0.98-1.19) when modeling PPD as a dichotomous outcome and using the sensitive PPD definition. HMW phthalates were associated with increased odds of PPD (OR, 1.11; 95% CI, 1.00-1.23 and OR, 1.10; 95% CI, 0.96-1.27) for the sensitive and specific PPD definitions, respectively. Sensitivity analyses produced stronger results. Conclusions and Relevance: Phthalates, ubiquitous chemicals in the environment, may be associated with PPD and could serve as important modifiable targets for preventive interventions. Future studies are needed to confirm these observations.

Intronic non-CG DNA hydroxymethylation and alternative mRNA splicing in honey bees.

BACKGROUND: Previous whole-genome shotgun bisulfite sequencing experiments showed that DNA cytosine methylation in the honey bee (Apis mellifera) is almost exclusively at CG dinucleotides in exons. However, the most commonly used method, bisulfite sequencing, cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine, an oxidized form of 5-methylcytosine that is catalyzed by the TET family of dioxygenases. Furthermore, some analysis software programs under-represent non-CG DNA methylation and hydryoxymethylation for a variety of reasons. Therefore, we used an unbiased analysis of bisulfite sequencing data combined with molecular and bioinformatics approaches to distinguish 5-methylcytosine from 5-hydroxymethylcytosine. By doing this, we have performed the first whole genome analyses of DNA modifications at non-CG sites in honey bees and correlated the effects of these DNA modifications on gene expression and alternative mRNA splicing. RESULTS: We confirmed, using unbiased analyses of whole-genome shotgun bisulfite sequencing (BS-seq) data, with both new data and published data, the previous finding that CG DNA methylation is enriched in exons in honey bees. However, we also found evidence that cytosine methylation and hydroxymethylation at non-CG sites is enriched in introns. Using antibodies against 5-hydroxmethylcytosine, we confirmed that DNA hydroxymethylation at non-CG sites is enriched in introns. Additionally, using a new technique, Pvu-seq (which employs the enzyme PvuRts1l to digest DNA at 5-hydroxymethylcytosine sites followed by next-generation DNA sequencing), we further confirmed that hydroxymethylation is enriched in introns at non-CG sites. CONCLUSIONS: Cytosine hydroxymethylation at non-CG sites might have more functional significance than previously appreciated, and in honey bees these modifications might be related to the regulation of alternative mRNA splicing by defining the locations of the introns.

Evidence for an epigenetic mechanism by which Hsp90 acts as a capacitor for morphological evolution.

Morphological alterations have been shown to occur in Drosophila melanogaster when function of Hsp90 (heat shock 90-kDa protein 1alpha, encoded by Hsp83) is compromised during development. Genetic selection maintains the altered phenotypes in subsequent generations. Recent experiments have shown, however, that phenotypic variation still occurs in nearly isogenic recombinant inbred strains of Arabidopsis thaliana. Using a sensitized isogenic D. melanogaster strain, iso-Kr(If-1), we confirm this finding and present evidence supporting an epigenetic mechanism for Hsp90's capacitor function, whereby reduced activity of Hsp90 induces a heritably altered chromatin state. The altered chromatin state is evidenced by ectopic expression of the morphogen wingless in eye imaginal discs and a corresponding abnormal eye phenotype, both of which are epigenetically heritable in subsequent generations, even when function of Hsp90 is restored. Mutations in nine different genes of the trithorax group that encode chromatin-remodeling proteins also induce the abnormal phenotype. These findings suggest that Hsp90 acts as a capacitor for morphological evolution through epigenetic and genetic mechanisms.

Epigenetic Reprogramming in Mice and Humans: From Fertilization to Primordial Germ Cell Development.

In this review, advances in the understanding of epigenetic reprogramming from fertilization to the development of primordial germline cells in a mouse and human embryo are discussed. To gain insights into the molecular underpinnings of various diseases, it is essential to comprehend the intricate interplay between genetic, epigenetic, and environmental factors during cellular reprogramming and embryonic differentiation. An increasing range of diseases, including cancer and developmental disorders, have been linked to alterations in DNA methylation and histone modifications. Global epigenetic reprogramming occurs in mammals at two stages: post-fertilization and during the development of primordial germ cells (PGC). Epigenetic reprogramming after fertilization involves rapid demethylation of the paternal genome mediated through active and passive DNA demethylation, and gradual demethylation in the maternal genome through passive DNA demethylation. The de novo DNA methyltransferase enzymes, Dnmt3a and Dnmt3b, restore DNA methylation beginning from the blastocyst stage until the formation of the gastrula, and DNA maintenance methyltransferase, Dnmt1, maintains methylation in the somatic cells. The PGC undergo a second round of global demethylation after allocation during the formative pluripotent stage before gastrulation, where the imprints and the methylation marks on the transposable elements known as retrotransposons, including long interspersed nuclear elements (LINE-1) and intracisternal A-particle (IAP) elements are demethylated as well. Finally, DNA methylation is restored in the PGC at the implantation stage including sex-specific imprints corresponding to the sex of the embryo. This review introduces a novel perspective by uncovering how toxicants and stress stimuli impact the critical period of allocation during formative pluripotency, potentially influencing both the quantity and quality of PGCs. Furthermore, the comprehensive comparison of epigenetic events between mice and humans breaks new ground, empowering researchers to make informed decisions regarding the suitability of mouse models for their experiments.

Pathological epigenetic events and reversibility review: the intersection between hallmarks of aging and developmental origin of health and disease.

We discuss pathological epigenetic events that are reversible (PEERs). A recent study by Poganik and colleagues showed that severe stress in mice and humans transiently elevates biological age of several tissues, and this transient age increase is reversible when the stress is removed. These studies suggest new strategies for reversing normal aging. However, it is important to note that developmental origin of health and disease studies have shown that developmental exposure to toxic chemicals such as lead causes permanent changes in neuron shape, connectivity and cellular hyperplasia of organs such as the heart and liver. In this review, the PEER hypothesis speculates that the hallmarks of aging and the hallmarks of developmental origin of health and disease intersect at PEERs.

The main goal in aging research is to find treatments to reverse aging. There are nine hallmarks of aging which describe cellular mechanisms that change as we age. For example, one of the hallmarks of aging is cellular senescence, which means that cells stop dividing when they get old. In this review, we describe nine hallmarks of developmental origins of health and disease (DOHaD). DOHaD studies show that exposures of the mother during pregnancy to stress or toxic chemicals can alter the health of the child throughout the child's lifespan. We argue that six of the nine hallmarks of DOHaD overlap with the hallmarks of aging and are reversible by dietary restriction or by drugs such as rapamycin which affect nutrient signaling. Based on this finding, we have formulated a hypothesis that we call 'pathological epigenetic events that are reversible' that contain the six hallmarks that overlap between the hallmarks of aging and the hallmarks of DOHaD. With this unexpected connection between aging and DOHaD, we argue that findings in one field, such as drugs that reverse aging, can apply to treatments in the other field, such as ways to reverse the adverse effects of exposures during pregnancy.

Biological Aging Acceleration Due to Environmental Exposures: An Exciting New Direction in Toxicogenomics Research.

Biological clock technologies are designed to assess the acceleration of biological age (B-age) in diverse cell types, offering a distinctive opportunity in toxicogenomic research to explore the impact of environmental stressors, social challenges, and unhealthy lifestyles on health impairment. These clocks also play a role in identifying factors that can hinder aging and promote a healthy lifestyle. Over the past decade, researchers in epigenetics have developed testing methods that predict the chronological and biological age of organisms. These methods rely on assessing DNA methylation (DNAm) levels at specific CpG sites, RNA levels, and various biomolecules across multiple cell types, tissues, and entire organisms. Commonly known as 'biological clocks' (B-clocks), these estimators hold promise for gaining deeper insights into the pathways contributing to the development of age-related disorders. They also provide a foundation for devising biomedical or social interventions to prevent, reverse, or mitigate these disorders. This review article provides a concise overview of various epigenetic clocks and explores their susceptibility to environmental stressors.

A single cell transcriptomic fingerprint of stressed premature, imbalanced differentiation of embryonic stem cells.

Cultured naïve pluripotent ESC differentiate into first lineage, XEN or second lineage, formative pluripotency. Hyperosmotic stress (sorbitol), like retinoic acid, decreases naive pluripotency and increases XEN in two ESC lines, as reported by bulk and scRNAseq, analyzed by UMAP. Sorbitol overrides pluripotency in two ESC lines as reported by bulk and scRNAseq, analyzed by UMAP. UMAP analyzed the effects of 5 stimuli - three stressed (200-300mM sorbitol with leukemia inhibitory factor +LIF) and two unstressed (+LIF, normal stemness-NS and -LIF, normal differentiation-ND). Sorbitol and RA decrease naive pluripotency and increase subpopulations of 2-cell embryo-like and XEN sub-lineages; primitive, parietal, and visceral endoderm (VE). Between the naïve pluripotency and primitive endoderm clusters is a stress-induced cluster with transient intermediate cells with higher LIF receptor signaling, with increased Stat3, Klf4, and Tbx3 expression. Sorbitol, like RA, also suppresses formative pluripotency, increasing lineage imbalance. Although bulk RNAseq and gene ontology group analyses suggest that stress induces head organizer and placental markers, scRNAseq reveals few cells. But VE and placental markers/cells were in adjacent clusters, like recent reports. UMAPs show that dose-dependent stress overrides stemness to force premature lineage imbalance. Hyperosmotic stress induces lineage imbalance, and other toxicological stresses, like drugs with RA, may cause lineage imbalance, resulting in miscarriages or birth defects.

Intrinsic sexual dimorphism in the placenta determines the differential response to benzene exposure.

Maternal immune activation (MIA) by environmental challenges is linked to severe developmental complications, such as neurocognitive disorders, autism, and even fetal/maternal death. Benzene is a major toxic compound in air pollution that affects the mother as well as the fetus and has been associated with reproductive complications. Our objective was to elucidate whether benzene exposure during gestation triggers MIA and its impact on fetal development. We report that benzene exposure during pregnancy leads MIA associated with increased fetal resorptions, fetal growth, and abnormal placenta development. Furthermore, we demonstrate the existence of a sexual dimorphic response to benzene exposure in male and female placentas. The sexual dimorphic response is a consequence of inherent differences between male and female placenta. These data provide crucial information on the origins or sexual dimorphism and how exposure to environmental factors can have a differential impact on the development of male and female offspring.

Evaluation of pediatric epigenetic clocks across multiple tissues.

BACKGROUND: Epigenetic clocks are promising tools for assessing biological age. We assessed the accuracy of pediatric epigenetic clocks in gestational and chronological age determination. RESULTS: Our study used data from seven tissue types on three DNA methylation profiling microarrays and found that the Knight and Bohlin clocks performed similarly for blood cells, while the Lee clock was superior for placental samples. The pediatric-buccal-epigenetic clock performed the best for pediatric buccal samples, while the Horvath clock is recommended for children's blood cell samples. The NeoAge clock stands out for its unique ability to predict post-menstrual age with high correlation with the observed age in infant buccal cell samples. CONCLUSIONS: Our findings provide valuable guidance for future research and development of epigenetic clocks in pediatric samples, enabling more accurate assessments of biological age.

10 Years of Toxicogenomics section in Frontiers in Genetics: Past discoveries and Future Perspectives.

The Frontiers Media family has over 200 journals, which are each headed by usually one Field Chief Editor, and several thousand specialty sections, which are each headed by one or more Specialty Chief Editors. The year 2021 was the 10th anniversary of the founding of the Frontiers in Genetics journal and the Frontiers in Toxicogenomics specialty section of this journal. In 2021, we also announce one of the newest of the Frontiers journals-Frontiers in Toxicology which is part of the Frontiers Media family of journals but independent of Frontiers in Genetics. Dr. Ruden is the founding, and currently sole, Specialty Chief Editor of Frontiers in Toxicogenomics and one of 9 Specialty Chief Editors of Frontiers in Toxicology. As of 2021, Frontiers in Toxicogenomics has published over 138 articles and has over 370 Editors including 90 Associate Editors and 280 Review Editors. The Frontiers in Genetics impact factor was initially approximately 2.5 when it was first listed in PubMed in 2015 and has risen steadily to its current value of 4.8, which is typical for the majority of the over 200 Frontiers journals that have established impact factors. In this overview of the first decade of Frontiers in Toxicogenomics, we discuss the top 5 articles with the highest Scopus citations, which were all written in the first few years of the journal. The article with the highest number of citations, with 353 Scopus over 600 Google Scholar citations, and the highest average number of citations (67) that steadily increased from 10 citations in 2013 to 119 citations in 2021, was written in 2012 by Dr. Ruden's laboratory and titled, "Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift." The five most influential authors who published in the journal in the past 10 years based on Scopus citations of a particular paper are Dr. Ruden's laboratory, with 353 Scopus citations for the SnpSift paper mentioned above; Drs. Brock Christensen and Carmen J. Marsit, with 86 Scopus citations for their review, "Epigenomics in environmental health"; Dr. Michael Aschner and colleagues, with 61 Scopus citations for their paper "Genetic factors and manganese-induced neurotoxicity"; and Dr. Sandra C. dos Santos and colleagues, with 59 Scopus citations for their paper, "Yeast toxicogenomics: genome-wide responses to chemical stresses with impact in environmental health, pharmacology, and biotechnology." While the top 5 papers were published in the early years of the journal, we will also discuss a more recent article published in 2018 on a comparison of RNA-seq and microarray methods by Dr. Michael Liguori's laboratory, "Comparison of RNA-Seq and Microarray Gene Expression Platforms for the Toxicogenomic Evaluation of Liver From Short-Term Rat Toxicity Studies," that far exceeds the number of downloads and views of all the other articles published in the first 10 years of the journal and will likely be a top cited paper in the second decade highlights of this journal. Finally, we discuss where the Frontiers in Toxicogenomics specialty journal and the Frontiers in Toxicology journal will go to advance the field of toxicogenomics, and more generally, toxicology, in the future.

Prenatal Exposures to Common Phthalates and Prevalent Phthalate Alternatives and Infant DNA Methylation at Birth.

Phthalates are a diverse group of chemicals used in consumer products. Because they are so widespread, exposure to these compounds is nearly unavoidable. Recently, growing scientific consensus has suggested that phthalates produce health effects in developing infants and children. These effects may be mediated through mechanisms related to the epigenome, the constellation of mitotically heritable chemical marks and small compounds that guide transcription and translation. The present study examined the relationship between prenatal, first-trimester exposure of seven phthalates and epigenetics in two pregnancy cohorts (n = 262) to investigate sex-specific alterations in infant blood DNA methylation at birth (cord blood or neonatal blood spots). Prenatal exposure to several phthalates was suggestive of association with altered DNA methylation at 4 loci in males (all related to ΣDEHP) and 4 loci in females (1 related to ΣDiNP; 2 related to BBzP; and 1 related to MCPP) at a cutoff of q < 0.2. Additionally, a subset of dyads (n = 79) was used to interrogate the relationships between two compounds increasingly used as substitutions for common phthalates (ΣDINCH and ΣDEHTP) and cord blood DNA methylation. ΣDINCH, but not ΣDEHTP, was suggestive of association with DNA methylation (q < 0.2). Together, these results demonstrate that prenatal exposure to both classically used phthalate metabolites and their newer alternatives is associated with sex-specific infant DNA methylation. Research and regulatory actions regarding this chemical class should consider the developmental health effects of these compounds and aim to avoid regrettable substitution scenarios in the present and future.

Using Live Imaging and Fluorescence Ubiquitinated Cell Cycle Indicator Embryonic Stem Cells to Distinguish G1 Cell Cycle Delays for General Stressors like Perfluoro-Octanoic Acid and Hyperosmotic Sorbitol or G2 Cell Cycle Delay for Mutagenic Stressors like Benzo(a)pyrene.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 µM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

Epigenetics: new insights into postoperative adhesion development.

BACKGROUND: The link between post-operative adhesion development and epigenetic modifications is important in understanding the mechanism behind their formation. The purpose of this study was to determine whether epigenetic differences exist between primary fibroblasts of normal peritoneum and adhesion tissues isolated from the same patient(s). METHODS: DNA from fibroblasts isolated from normal peritoneum and adhesion tissues was isolated using Qiagen's EZ1 Advanced Kit. Methylation patterns of genes were quantified and compared in both cell lines using the Infinium Human Methylation 27 Beadchip system. RESULTS: A total of 7364 genes had been found to manifest significantly different DNA methylation levels in adhesion fibroblasts as compared to normal peritoneal fibroblasts (p<0.01). A total of 1685 genes were found to have increased DNA methylation by 50% in adhesion compared to peritoneal fibroblasts, and were enriched in Gene Ontology categories, Glycoprotein, and Defense Response. Furthermore, 1287 genes were found to have decreased DNA methylation patterns with enriched Gene Ontology categories, "Homeobox", and Transcription Factor Activity in adhesion fibroblasts. CONCLUSIONS: Epigenetic differences in fibroblasts isolated from normal peritoneum and adhesion tissues were observed. Future studies focusing on the precise role of these genes in the development of post operative adhesions will allow us to more fully appreciate regulatory mechanisms leading to adhesion development, thereby establishing targets for therapeutic interventions to prevent or limit adhesion development.

Using high throughput screens to predict miscarriages with placental stem cells and long-term stress effects with embryonic stem cells.

A problem in developmental toxicology is the massive loss of life from fertilization through gastrulation, and the surprising lack of knowledge of causes of miscarriage. Half to two-thirds of embryos are lost, and environmental and genetic causes are nearly equal. Simply put, it can be inferred that this is a difficult period for normal embryos, but that environmental stresses may cause homeostatic responses that move from adaptive to maladaptive with increasing exposures. At the lower 50% estimate, miscarriage causes greater loss-of-life than all cancers combined or of all cardio- and cerebral-vascular accidents combined. Surprisingly, we do not know if miscarriage rates are increasing or decreasing. Overshadowed by the magnitude of miscarriages, are insufficient data on teratogenic or epigenetic imbalances in surviving embryos and their stem cells. Superimposed on the difficult normal trajectory for peri-gastrulation embryos are added malnutrition, hormonal, and environmental stresses. An overarching hypothesis is that high throughput screens (HTS) using cultured viable reporter embryonic and placental stem cells (e.g., embryonic stem cells [ESC] and trophoblast stem cells [TSC] that report status using fluorescent reporters in living cells) from the pre-gastrulation embryo will most rapidly test a range of hormonal, environmental, nutritional, drug, and diet supplement stresses that decrease stem cell proliferation and imbalance stemness/differentiation. A second hypothesis is that TSC respond with greater sensitivity in magnitude to stress that would cause miscarriage, but ESC are stress-resistant to irreversible stemness loss and are best used to predict long-term health defects. DevTox testing needs more ESC and TSC HTS to model environmental stresses leading to miscarriage or teratogenesis and more research on epidemiology of stress and miscarriage. This endeavor also requires a shift in emphasis on pre- and early gastrulation events during the difficult period of maximum loss by miscarriage.