Putative Early Life Epigenetic Biomarkers of Hepatocellular Carcinoma in Mice Perinatally Exposed to Bisphenol A.

Bisphenol A (BPA) is a high production-volume chemical with hormone-like properties that has been implicated as a potential carcinogen. Early life exposure has been linked to increased risk for precancerous lesions in mammary and prostate glands and the uterus, but no prior study has shown a significant association between cancer development and exposure to BPA alone. The overall goal of this dissertation was to test the central hypothesis that early life BPA exposure dysregulates the DNA methylome and thereby modifies risk for adult liver tumors. Chapter 2 describes a monotonic increase in hepatic tumors with increasing dose of perinatal BPA. Chapter 2 further characterizes the observed liver tumor phenotype in a murine model and notes a lack of sexual dimorphism in incidence, as well as a lack of regenerative response to injury, suggesting a solely proliferative response to BPA. Chapter 3 provides proof of principle for a novel method for identification of epigenetic biomarkers of exposure and outcome across the life-course and across species. Chapter 4 demonstrates that epigenome-wide discovery experiments in animal models are effective tools for identification and understanding of paralagous epimutations in cell signaling pathways salient to human disease. Taken together, these findings are indicators of the relevance of the hepatic tumor phenotype seen in BPA-exposed mice to human health. This work combines a state-of-the-art epigenomic discovery approach with high resolution, quantitative epigenetic techniques to identify dose-dependent alterations in the fetal and adult epigenomes that correlate with HCC status. As such, this research represents a critical link between early life environment and a specific phenotypic outcome in later life, necessary to the determination of human health risk assessment and human disease prevention, diagnosis, and treatmentPhDEnvironmental Health SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111610/1/carenw_3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111610/2/carenw_2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/111610/3/carenw_1.pd

Epigenome‐wide DNA methylation analysis implicates neuronal and inflammatory signaling pathways in adult murine hepatic tumorigenesis following perinatal exposure to bisphenol A

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/133545/1/em22024.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133545/2/em22024_am.pd

Bisphenol A-associated epigenomic changes in prepubescent girls: a cross-sectional study in Gharbiah, Egypt

Abstract Background There is now compelling evidence that epigenetic modifications link adult disease susceptibility to environmental exposures during specific life stages, including pre-pubertal development. Animal studies indicate that bisphenol A (BPA), the monomer used in epoxy resins and polycarbonate plastics, may impact health through epigenetic mechanisms, and epidemiological data associate BPA levels with metabolic disorders, behavior changes, and reproductive effects. Thus, we conducted an environmental epidemiology study of BPA exposure and CpG methylation in pre-adolescent girls from Gharbiah, Egypt hypothesizing that methylation profiles exhibit exposure-dependent trends. Methods Urinary concentrations of total (free plus conjugated) species of BPA in spot samples were quantified for 60 girls aged 10 to 13. Genome-wide CpG methylation was concurrently measured in bisulfite-converted saliva DNA using the Infinium HumanMethylation27 BeadChip (N = 46). CpG sites from four candidate genes were validated via quantitative bisulfite pyrosequencing. Results CpG methylation varied widely among girls, and higher urinary BPA concentrations were generally associated with less genomic methylation. Based on pathway analyses, genes exhibiting reduced methylation with increasing urinary BPA were involved in immune function, transport activity, metabolism, and caspase activity. In particular, hypomethylation of CpG targets on chromosome X was associated with higher urinary BPA. Using the Comparative Toxicogenomics Database, we identified a number of candidate genes in our sample that previously have been associated with BPA-related expression change. Conclusions These data indicate that BPA may affect human health through specific epigenomic modification of genes in relevant pathways. Thus, epigenetic epidemiology holds promise for the identification of biomarkers from previous exposures and the development of epigenetic-based diagnostic strategies.http://deepblue.lib.umich.edu/bitstream/2027.42/112909/1/12940_2013_Article_648.pd

CoNaMad-Cohorte de Nacimiento de Madre de Dios/Madre de Dios Birth Cohort to Study Effects of in-utero Trace Metals Exposure in the Southern Peruvian Amazon.

Background: In-utero exposure to mercury and other trace metals pose a significant threat to child health and development, but exposures and health impacts in artisanal and small-scale gold mining (ASGM) environments are poorly defined. Objectives: We describe the CONAMAD study design, a prospective birth cohort consisting of multiparous women (18 and over) living in rural and peri-urban Peruvian Amazon communities exposed to ASGM. Methods: Pregnant women are enrolled from health posts across four zones of Madre de Dios, Peru. Data are collected at enrollment, childbirth, and (planned) 36-48 months. At enrollment, hair samples for mercury assessment, demographic and clinical data are obtained. At birth, we obtain venous and cord blood, placenta, hair, toenails, and saliva. Findings: Two hundred seventy mothers were enrolled at an average 20 weeks gestational age with no differences in maternal characteristics across zones. Two hundred fifteen mothers were successfully followed at birth. We obtained 214 maternal and cord blood samples, 211 maternal and 212 infant hair samples, 212 placenta samples, 210 infant saliva samples, and 214 infant dried blood spots. Data collected will allow for testing our primary hypotheses of maternal malnutrition modifying ratios of cord:maternal blood total mercury (tHg), cord blood:maternal hair tHg, and infant:maternal hair tHg, and whether chemical mixtures (Hg, Pb, Cd) have synergistic effects on infant neurodevelopment. Conclusions: CONAMAD is designed to collect and store samples for future processing and hypothesis testing associated with in-utero mercury exposure and child development. We have completed the exposure assessments and will conduct a follow-up of mothers to evaluate early child development outcomes, including developmental delay and growth. These data offer insights into disease mechanisms, exposure prevention, and policy guidance for countries where ASGM is prevalent

<i>Stat3</i> is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

<p>Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (<i>Esr1, Il-6st</i>, and <i>Stat3</i>) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, <i>Stat3</i>, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating <i>Stat3</i> as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within <i>STAT3</i> varied with liver tissue BPA level in human fetal liver samples as well, suggesting <i>STAT3</i> may be a translationally relevant candidate biomarker. These data implicate <i>Stat3</i> as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.</p

Adaptive radiation-induced epigenetic alterations mitigated by antioxidants

Humans are exposed to low-dose ionizing radiation (LDIR) from a number of environmental and medical sources. In addition to inducing genetic mutations, there is concern that LDIR may also alter the epigenome. Such heritable effects early in life can either be positively adaptive or result in the enhanced formation of diseases, including cancer, diabetes, and obesity. Herein, we show that LDIR significantly increased DNA methylation at the viable yellow agouti (A(vy)) locus in a sex-specific manner (P=0.004). Average DNA methylation was significantly increased in male offspring exposed to doses between 0.7 and 7.6 cGy, with maximum effects at 1.4 and 3.0 cGy (P<0.01). Offspring coat color was concomitantly shifted toward pseudoagouti (P<0.01). Maternal dietary antioxidant supplementation mitigated both the DNA methylation changes and coat color shift in the irradiated offspring. Thus, LDIR exposure during gestation elicits epigenetic alterations that lead to positive adaptive phenotypic changes that are negated with antioxidants, indicating they are mediated in part by oxidative stress. These findings provide evidence that in the isogenic A(vy) mouse model, epigenetic alterations resulting from LDIR play a role in radiation hormesis, bringing into question the assumption that every dose of radiation is harmful

Variable histone modifications at the Avy metastable epiallele

The ability of environmental factors to shape health and disease involves epigenetic mechanisms that mediate gene-environment interactions. Metastable epiallele genes are variably expressed in genetically identical individuals due to epigenetic modifications established during early development. DNA methylation within metastable epialleles is stochastic due to probabilistic reprogramming of epigenetic marks during embryogenesis. Maternal nutrition and environment have been shown to affect metastable epiallele methylation patterns and subsequent adult phenotype. Little is known, however, about the role of histone modifications in influencing metastable epiallele expression and phenotypic variation. Utilizing chromatin immunoprecipitation followed by qPCR, we observe variable histone patterns in the 5′ long terminal repeat (LTR) of the murine viable yellow agouti (Avy) metastable epiallele. This region contains 6 CpG sites, which are variably methylated in isogenic Avy/a offspring. Yellow mice, which are hypomethylated at the Avy LTR and exhibit constitutive ectopic expression of Agouti (a), also display enrichment of H3 and H4 di-acetylation (p = 0.08 and 0.09, respectively). Pseudoagouti mice, in which Avy hypermethylation is thought to silence ectopic expression, exhibit enrichment of H4K20 tri-methylation (p = 0.01). No differences are observed for H3K4 tri-methylation (p = 0.7), a modification often enriched in the promoter of active genes. These results show for the first time the presence of variable histone modifications at a metastable epiallele, indicating that DNA methylation acts in concert with histone modifications to affect inter-individual variation of metastable epiallele expression. Therefore, the potential for environmental factors to influence histone modifications, in addition to DNA methylation, should be addressed in environmental epigenomic studies