Julia E. Rager

Epigenetic Changes in Individuals with Arsenicosis

Inorganic arsenic (iAs) is an environmental toxicant currently poisoning millions of people worldwide, and chronically exposed individuals are susceptible to arsenicosis or arsenic poisoning. Using a state-of-the-art technique to map the methylomes of our study subjects, we identified a large interactome of hypermethylated genes that are enriched for their involvement in arsenic-associated diseases, such as cancer, heart disease, and diabetes. Notably, we have uncovered an arsenic-induced tumor suppressorome, a complex of 17 tumor suppressors known to be silenced in human cancers. This finding represents a pivotal clue in unraveling a possible epigenetic mode of arsenic-induced disease.

Prenatal arsenic exposure and the epigenome: altered microRNAs associated with innate and adaptive immune signaling in newborn cord blood.

The Biomarkers of Exposure to ARsenic (BEAR) pregnancy cohort in Gómez Palacio, Mexico was recently established to better understand the impacts of prenatal exposure to inorganic arsenic (iAs). In this study, we examined a subset (n = 40) of newborn cord blood samples for microRNA (miRNA) expression changes associated with in utero arsenic exposure. Levels of iAs in maternal drinking water (DW‐iAs) and maternal urine were assessed. Levels of DW‐iAs ranged from below detectable values to 236 µg/L (mean = 51.7 µg/L). Total arsenic in maternal urine (U‐tAs) was defined as the sum of iAs and its monomethylated and dimethylated metabolites (MMAs and DMAs, respectively) and ranged from 6.2 to 319.7 µg/L (mean = 64.5 µg/L). Genome‐wide miRNA expression analysis of cord blood revealed 12 miRNAs with increasing expression associated with U‐tAs. Transcriptional targets of the miRNAs were computationally predicted and subsequently assessed using transcriptional profiling. Pathway analysis demonstrated that the U‐tAs‐associated miRNAs are involved in signaling pathways related to known health outcomes of iAs exposure including cancer and diabetes mellitus. Immune response‐related mRNAs were also identified with decreased expression levels associated with U‐tAs, and predicted to be mediated in part by the arsenic‐responsive miRNAs. Results of this study highlight miRNAs as novel responders to prenatal arsenic exposure that may contribute to associated immune response perturbations. Environ. Mol. Mutagen. 55:196–208, 2014.

Cadmium exposure and the epigenome: Exposure-associated patterns of DNA methylation in leukocytes from mother-baby pairs

Cadmium (Cd) is prevalent in the environment yet understudied as a developmental toxicant. Cd partially crosses the placental barrier from mother to fetus and is linked to detrimental effects in newborns. Here we examine the relationship between levels of Cd during pregnancy and 5-methylcytosine (5mC) levels in leukocyte DNA collected from 17 mother-newborn pairs. The methylation of cytosines is an epigenetic mechanism known to impact transcriptional signaling and influence health endpoints. A methylated cytosine-guanine (CpG) island recovery assay was used to assess over 4.6 million sites spanning 16,421 CpG islands. Exposure to Cd was classified for each mother-newborn pair according to maternal blood levels and compared with levels of cotinine. Subsets of genes were identified that showed altered DNA methylation levels in their promoter regions in fetal DNA associated with levels of Cd (n = 61), cotinine (n = 366), or both (n = 30). Likewise, in maternal DNA, differentially methylated genes were identified that were associated with Cd (n = 92) or cotinine (n = 134) levels. While the gene sets were largely distinct between maternal and fetal DNA, functional similarities at the biological pathway level were identified including an enrichment of genes that encode for proteins that control transcriptional regulation and apoptosis. Furthermore, conserved DNA motifs with sequence similarity to specific transcription factor binding sites were identified within the CpG islands of the gene sets. This study provides evidence for distinct patterns of DNA methylation or “footprints” in fetal and maternal DNA associated with exposure to Cd.

Formaldehyde Carcinogenicity Research 30 Years and Counting for Mode of Action, Epidemiology, and Cancer Risk Assessment

Formaldehyde is a widely used high production chemical that is also released as a byproduct of combustion, off-gassing of various building products, and as a fixative for pathologists and embalmers. What is not often realized is that formaldehyde is also produced as a normal physiologic chemical in all living cells. In 1980, chronic inhalation of high concentrations of formaldehyde was shown to be carcinogenic, inducing a high incidence of nasal squamous cell carcinomas in rats. Some epidemiologic studies have also found increased numbers of nasopharyngeal carcinoma and leukemia in humans exposed to formaldehyde that resulted in formaldehyde being considered a Known Human Carcinogen. This article reviews the data for rodent and human carcinogenicity, early Mode of Action studies, more recent molecular studies of both endogenous and exogenous DNA adducts, and epigenetic studies. It goes on to demonstrate the power of these research studies to provide critical data to improve our ability to develop science-based cancer risk assessments, instead of default approaches. The complexity of constant physiologic exposure to a known carcinogen requires that new ways of thinking be incorporated into determinations of cancer risk assessment for formaldehyde, other endogenous carcinogens, and the role of background endogenous DNA damage and mutagenesis.

Arsenic and the Epigenome: Interindividual Differences in Arsenic Metabolism Related to Distinct Patterns of DNA Methylation

Biotransformation of inorganic arsenic (iAs) is one of the factors that determines the character and magnitude of the diverse detrimental health effects associated with chronic iAs exposure, but it is unknown how iAs biotransformation may impact the epigenome. Here, we integrated analyses of genome‐wide, gene‐specific promoter DNA methylation levels of peripheral blood leukocytes with urinary arsenical concentrations of subjects from a region of Mexico with high levels of iAs in drinking water. These analyses revealed dramatic differences in DNA methylation profiles associated with concentrations of specific urinary metabolites of arsenic (As). The majority of individuals in this study had positive indicators of As‐related disease, namely pre‐diabetes mellitus or diabetes mellitus (DM). Methylation patterns of genes with known associations with DM were associated with urinary concentrations of specific iAs metabolites. Future studies will determine whether these DNA methylation profiles provide mechanistic insight into the development of iAs‐associated disease, predict disease risk, and/or serve as biomarkers of iAs exposure in humans.

Epigenetic Changes Induced by Air Toxics: Formaldehyde Exposure Alters miRNA Expression Profiles in Human Lung Cells

Background Exposure to formaldehyde, a known air toxic, is associated with cancer and lung disease. Despite the adverse health effects of formaldehyde, the mechanisms underlying formaldehyde-induced disease remain largely unknown. Research has uncovered microRNAs (miRNAs) as key posttranscriptional regulators of gene expression that may influence cellular disease state. Although studies have compared different miRNA expression patterns between diseased and healthy tissue, this is the first study to examine perturbations in global miRNA levels resulting from formaldehyde exposure. Objectives We investigated whether cellular miRNA expression profiles are modified by formaldehyde exposure to test the hypothesis that formaldehyde exposure disrupts miRNA expression levels within lung cells, representing a novel epigenetic mechanism through which formaldehyde may induce disease. Methods Human lung epithelial cells were grown at air–liquid interface and exposed to gaseous formaldehyde at 1 ppm for 4 hr. Small RNAs and protein were collected and analyzed for miRNA expression using microarray analysis and for interleukin (IL-8) protein levels by enzyme-linked immunosorbent assay (ELISA). Results Gaseous formaldehyde exposure altered the miRNA expression profiles in human lung cells. Specifically, 89 miRNAs were significantly down-regulated in formaldehyde-exposed samples versus controls. Functional and molecular network analysis of the predicted miRNA transcript targets revealed that formaldehyde exposure potentially alters signaling pathways associated with cancer, inflammatory response, and endocrine system regulation. IL-8 release increased in cells exposed to formaldehyde, and results were confirmed by real-time polymerase chain reaction. Conclusions Formaldehyde alters miRNA patterns that regulate gene expression, potentially leading to the initiation of a variety of diseases.

17β-Estradiol and Tamoxifen Prevent Gastric Cancer by Modulating Leukocyte Recruitment and Oncogenic Pathways in Helicobacter Pylori–Infected INS-GAS Male Mice

Helicobacter pylori infection promotes male predominant gastric adenocarcinoma in humans. Estrogens reduce gastric cancer risk and previous studies showed that prophylactic 17β-estradiol (E2) in INS-GAS mice decreases H. pylori–induced carcinogenesis. We examined the effect of E2 and tamoxifen (TAM) on H. pylori–induced gastric cancer in male and female INS-GAS mice. After confirming robust gastric pathology at 16 weeks postinfection (WPI), mice were implanted with E2, TAM, both E2 and TAM, or placebo pellets for 12 weeks. At 28 WPI, gastric histopathology, gene expression, and immune cell infiltration were evaluated and serum inflammatory cytokines measured. After treatment, no gastric cancer was observed in H. pylori–infected males receiving E2 and/or TAM, whereas 40% of infected untreated males developed gastric cancer. E2, TAM, and their combination significantly reduced gastric precancerous lesions in infected males compared with infected untreated males (P < 0.001, 0.01, and 0.01, respectively). However, TAM did not alter female pathology regardless of infection status. Differentially expressed genes from males treated with E2 or TAM (n = 363 and n = 144, Q < 0.05) associated highly with cancer and cellular movement, indicating overlapping pathways in the reduction of gastric lesions. E2 or TAM deregulated genes associated with metastasis (PLAUR and MMP10) and Wnt inhibition (FZD6 and SFRP2). Compared with controls, E2 decreased gastric mRNA (Q < 0.05) and serum levels (P < 0.05) of CXCL1, a neutrophil chemokine, leading to decreased neutrophil infiltration (P < 0.01). Prevention of H. pylori–induced gastric cancer by E2 and TAM may be mediated by estrogen signaling and is associated with decreased CXCL1, decreased neutrophil counts, and downregulation of oncogenic pathways. Cancer Prev Res; 4(9); 1426–35. ©2011 AACR.

Air toxics and epigenetic effects: ozone altered microRNAs in the sputum of human subjects

Ozone (O3) is a criteria air pollutant that is associated with numerous adverse health effects, including altered respiratory immune responses. Despite its deleterious health effects, possible epigenetic mechanisms underlying O3-induced health effects remain understudied. MicroRNAs (miRNAs) are epigenetic regulators of genomic response to environmental insults and unstudied in relationship to O3 inhalation exposure. Our objective was to test whether O3 inhalation exposure significantly alters miRNA expression profiles within the human bronchial airways. Twenty healthy adult human volunteers were exposed to 0.4 ppm O3 for 2 h. Induced sputum samples were collected from each subject 48 h preexposure and 6 h postexposure for evaluation of miRNA expression and markers of inflammation in the airways. Genomewide miRNA expression profiles were evaluated by microarray analysis, and in silico predicted mRNA targets of the O3-responsive miRNAs were identified and validated against previously measured O3-induced changes in mRNA targets. Biological network analysis was performed on the O3-associated miRNAs and mRNA targets to reveal potential associated response signaling and functional enrichment. Expression analysis of the sputum samples revealed that O3 exposure significantly increased the expression levels of 10 miRNAs, namely miR-132, miR-143, miR-145, miR-199a*, miR-199b-5p, miR-222, miR-223, miR-25, miR-424, and miR-582-5p. The miRNAs and their predicted targets were associated with a diverse range of biological functions and disease signatures, noted among them inflammation and immune-related disease. The present study shows that O3 inhalation exposure disrupts select miRNA expression profiles that are associated with inflammatory and immune response signaling. These findings provide novel insight into epigenetic regulation of responses to O3 exposure.

The epigenetic effects of a high prenatal folate intake in male mouse fetuses exposed in utero to arsenic.

Inorganic arsenic (iAs) is a complete transplacental carcinogen in mice. Previous studies have demonstrated that in utero exposure to iAs promotes cancer in adult mouse offspring, possibly acting through epigenetic mechanisms. Humans and rodents enzymatically convert iAs to its methylated metabolites. This reaction requires S-adenosylmethionine (SAM) as methyl group donor. SAM is also required for DNA methylation. Supplementation with folate, a major dietary source of methyl groups for SAM synthesis, has been shown to modify iAs metabolism and the adverse effects of iAs exposure. However, effects of gestational folate supplementation on iAs metabolism and fetal DNA methylation have never been thoroughly examined. In the present study, pregnant CD1 mice were fed control (i.e. normal folate, or 2.2 mg/kg) or high folate diet (11 mg/kg) from gestational day (GD) 5 to 18 and drank water with 0 or 85 ppm of As (as arsenite) from GD8 to 18. The exposure to iAs significantly decreased body weight of GD18 fetuses and increased both SAM and S-adenosylhomocysteine (SAH) concentrations in fetal livers. High folate intake lowered the burden of total arsenic in maternal livers but did not prevent the effects of iAs exposure on fetal weight or hepatic SAM and SAH concentrations. In fact, combined folate-iAs exposure caused further significant body weight reduction. Notably, iAs exposure alone had little effect on DNA methylation in fetal livers. In contrast, the combined folate-iAs exposure changed the CpG island methylation in 2,931 genes, including genes known to be imprinted. Most of these genes were associated with neurodevelopment, cancer, cell cycle, and signaling networks. The canonical Wnt-signaling pathway, which regulates fetal development, was among the most affected biological pathways. Taken together, our results suggest that a combined in utero exposure to iAs and a high folate intake may adversely influence DNA methylation profiles and weight of fetuses, compromising fetal development and possibly increasing the risk for early-onset of disease in offspring.

Influenza enhances caspase-1 in bronchial epithelial cells from asthmatic volunteers and is associated with pathogenesis

BACKGROUND The leading cause of asthma exacerbation is respiratory viral infection. Innate antiviral defense pathways are altered in the asthmatic epithelium, yet involvement of inflammasome signaling in virus-induced asthma exacerbation is not known. OBJECTIVE This study compared influenza-induced activation of inflammasome and innate immune signaling in human bronchial epithelial cells from volunteers with and without asthma and investigated the role of caspase-1 in epithelial cell antiviral defense. METHODS Differentiated primary human bronchial epithelial cells from volunteers with and without asthma were infected with influenza A virus. An inflammasome-specific quantitative real-time polymerase chain reaction array was used to compare baseline and influenza-induced gene expression profiles. Cytokine secretion, innate immune gene expression, and viral replication were compared between human bronchial epithelial cells from volunteers with and without asthma. Immunofluorescence microscopy was used to evaluate caspase-1 and PYCARD colocalization. Tracheal epithelial cells from caspase-1-deficient or wild-type mice were infected with influenza and assessed for antiviral gene expression and viral replication. RESULTS Human bronchial epithelial cells from asthmatic volunteers had altered influenza-induced expression of inflammasome-related and innate immune signaling components, which correlated with enhanced production of IL-1β, IL-6, and TNF-α. Specifically, influenza-induced caspase-1 expression was enhanced and localization differed in human bronchial epithelial cells from asthmatic volunteers compared to volunteers without asthma. Influenza-infected tracheal epithelial cells from caspase-1-deficient mice had reduced expression of antiviral genes and viral replication. CONCLUSION Caspase-1 plays an important role in the airway epithelial cell response to influenza infection, which is enhanced in asthmatic volunteers, and may contribute to the enhanced influenza-related pathogenesis observed in vivo.