Identification of a unique gene expression signature in mercury and 2,3,7,8-tetrachlorodibenzo-p-dioxin co-exposed cells.
ABSTRACT: Mercury (Hg) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are major environmental contaminants that commonly co-occur in the environment. Both Hg and TCDD are associated with a number of human diseases including cancers. While the individual toxicological effects of Hg and TCDD have been extensively investigated, studies on co-exposure are limited to a few genes and pathways. Therefore, a significant knowledge gap exists in the understanding of the deleterious effects of co-exposure to Hg and TCDD. Due to the prevalence of Hg and TCDD co-contamination in the environment and the major human health hazards they pose, it is important to obtain a fuller understanding of genome-wide effects of Hg and TCDD co-exposure. In this study, by performing a comprehensive transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) exposed to Hg and TCDD individually and in combination, we have uncovered a subset of genes with altered expression only in the co-exposed cells. We also identified the additive as well as antagonistic effects of Hg and TCDD on gene expression. Moreover, we found that co-exposure impacted several biological and disease processes not affected by Hg or TCDD individually. Our studies show that the consequences of Hg and TCDD co-exposure on the transcriptional program and biological processes could be substantially different from single exposures, thus providing new insights into the co-exposure-specific pathogenic processes.
Project description:Mercury (Hg) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are major environmental contaminants that commonly co-occur in the environment. Both Hg and TCDD are associated with a number of human diseases including cancers. While the individual toxicological effects of Hg and TCDD have been extensively investigated, studies on co-exposure are limited to a few genes and pathways. Therefore, a significant knowledge gap exists in the understanding of the deleterious effects of co-exposure to Hg and TCDD. Due to the prevalence of Hg and TCDD co-contamination in the environment and the major human health hazards they pose, it is important to obtain a fuller understanding of genome-wide effects of Hg and TCDD co-exposure. In this study, by performing a comprehensive transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) exposed to Hg and TCDD individually and in combination, we have uncovered a subset of genes with altered expression only in the co-exposed cells. We also identified the additive as well as antagonistic effects of Hg and TCDD on gene expression. Moreover, we found that co-exposure impacted several biological and disease processes not affected by Hg or TCDD individually. Our studies show that the consequences of Hg and TCDD co-exposure on the transcriptional program and biological processes could be substantially different from single exposures, thus providing new insights into the co-exposure-specific pathogenic processes. Overall design: Methods: we performed a comprehensive analysis of the transcriptomes of cells exposed to Hg or TCDD individually and in combination. RNA Seq gene expression comparison done in replicates.
Project description:Cardiovascular malformations are one of the most common congenital birth defects observed in humans. Defects in cardiac valves disrupt normal blood flow. Zebrafish are an outstanding experimental model for studying the effects that environmental contaminants have on developmental processes. Previous research has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes blood regurgitation in the heart and reduces peripheral blood flow in embryonic zebrafish, suggesting some form of valve failure. To test this we used video microscopy to examine valve function and structure in developing zebrafish exposed to TCDD. TCDD exposure produced blood regurgitation at both the atrioventricular (AV) and bulboventricular (BV) junctions. In marked contrast to control embryos exposed to the vehicle dimethyl sulfoxide, embryos exposed to TCDD failed to form valve leaflets as the heart matured. In addition, whereas TCDD did not block initial formation of the bulbus arteriosus, we found that TCDD exposure prevented the normal growth and development of this portion of the outflow tract. TCDD altered the localization of endothelial cells at the AV and BV junctions and altered the localized expression of mRNAs bmp4 and notch1b normally associated with the nascent valves. Taken together, our results demonstrate that although TCDD does not prevent the initial specification of the presumptive valve locations, TCDD exposure produces severe alterations in valve development, leading to blood regurgitation and failing circulation in the developing zebrafish.
Project description:Vertebrate jaw development can be disrupted by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-a potent activator of the aryl hydrocarbon receptor (AHR) transcription factor required for transducing the toxic effects of TCDD. We used zebrafish (Danio rerio) embryos to investigate transcriptional responses to TCDD with the goal of discovering novel, jaw-specific genes affected by TCDD exposure. Our results uncovered a novel target of TCDD-activated Ahr belonging to the evolutionarily conserved family of forkhead box transcription factors. Quantitative real-time polymerase chain reaction analysis demonstrated that FoxQ1b was upregulated by TCDD 7- and 10-fold at 24 and 48 h postfertilization (hpf), respectively. The rate of TCDD-induced FoxQ1b expression was more rapid than that of Cyp1a, a known direct target of TCDD-activated Ahr. TCDD-mediated induction of FoxQ1b was suppressed in the presence of an Ahr antagonist, alpha-naphthoflavone, as well as following knockdown of Ahr2 expression using an Ahr2-specific morpholino antisense oligonucleotide. In situ hybridization analysis of FoxQ1b expression at 48 hpf demonstrated that FoxQ1b is specifically expressed in the jaw primordium where it discretely outlines a developing jaw structure known as Meckel's cartilage--a conserved structure in all jawed vertebrates that develops abnormally in the presence of TCDD. These results identify a novel target of TCDD-activated Ahr and suggest that FoxQ1b may play a role in craniofacial abnormalities induced by developmental exposure to TCDD.
Project description:Background:Long non-coding RNAs (lncRNAs) may regulate gene expression in numerous biological processes including cellular response to xenobiotics. The exposure of living organisms to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, results in reproductive defects in many species including pigs. The aims of the study were to identify and characterize lncRNAs in porcine granulosa cells as well as to examine the effects of TCDD on the lncRNA expression profile in the cells. Results:One thousand six hundred sixty-six lncRNAs were identified and characterized in porcine granulosa cells. The identified lncRNAs were found to be shorter than mRNAs. In addition, the number of exons was lower in lncRNAs than in mRNAs and their exons were longer. TCDD affected the expression of 22 lncRNAs (differentially expressed lncRNAs [DELs]; log2 fold change ???1, P-adjusted <?0.05) in the examined cells. Potential functions of DELs were indirectly predicted via searching their target cis- and trans-regulated protein-coding genes. The co-expression analysis revealed that DELs may influence the expression of numerous genes, including those involved in cellular response to xenobiotics, dioxin metabolism, endoplasmic reticulum stress and cell proliferation. Aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1) were found among the trans-regulated genes. Conclusions:These findings indicate that the identified lncRNAs may constitute a part of the regulatory mechanism of TCDD action in granulosa cells. To our knowledge, this is the first study describing lncRNAs in porcine granulosa cells as well as TCDD effects on the lncRNA expression profile. These results may trigger new research directions leading to better understanding of molecular processes induced by xenobiotics in the ovary.
Project description:Alternative splicing is a co-transcriptional mechanism by including or excluding exons in different combinations, thereby expanding the diversity of protein isoforms of a single gene. Abnormalities in this process can result in deleterious effects to human health, and several xenobiotics are known to interfere with splicing regulation through multiple mechanisms. These changes could lead to human diseases such as cancer, neurological disorders, autoimmune diseases, and developmental disorders. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant generated as a byproduct of various industrial activities. Exposure to this dioxin has been linked to a wide range of pathologies through the alteration of multiple cellular processes. However, the effects of TCDD exposure on alternative splicing have not yet been studied. Here, we investigate whether exposure to TCDD influenced hepatic alternative splicing in adult male C57BL/6J mice. We identified several genes whose alternative splicing of precursor messenger RNAs was modified following TCDD exposure. In particular, we demonstrated that alternative splicing of Cyp1a1, Ahrr, and Actn1 was significantly altered after TCDD treatment. These findings show that exposure to TCDD has an impact on alternative splicing, presumably through binding to the aryl hydrocarbon receptor, and suggest a new avenue for AhR-mediated pathogenesis. Overall design: Adult male C57BL/6 mice were treated by gavage with 0, 5, or 500 µg/kg TCDD dissolved in corn oil vehicle. Mice were euthanized 19 hours following treatment and liver tissue was harvested for analysis. RNA was isolated and the transcriptome for each animal assayed on separate microarrays.
Project description:2,3,7,8-tetrachlorodibenzo-p-dixion (TCDD) is the most potent of the dioxin congeners, capable of causing a wide range of toxic effects across numerous animal models. Previous studies have demonstrated that males and females of the same species can display divergent sensitivity phenotypes to TCDD toxicities. Although it is now clear that most TCDD-induced toxic outcomes are mediated by the aryl hydrocarbon receptor (AHR), the mechanism of differential responses to TCDD exposure between sexes remains largely unknown. To investigate the differential sensitivities in male and female mice, we profiled the hepatic transcriptomic responses 4 days following exposure to various amounts of TCDD (125, 250, 500 or 1000 ?g/kg) in adult male and female C57BL/6Kuo mice.Several key findings were revealed by our study. 1) Hepatic transcriptomes varied significantly between the sexes at all doses examined. 2) The liver transcriptome of males was more dysregulated by TCDD than that of females. 3) The alteration of "AHR-core" genes was consistent in magnitude, regardless of sex. 4) A subset of genes demonstrated sex-dependent TCDD-induced transcriptional changes, including Fmo3 and Nr1i3, which were significantly induced in livers of male mice only. In addition, a meta-analysis was performed to contrast transcriptomic profiles of various organisms and tissues following exposure to equitoxic doses of TCDD. Minimal overlap was observed in the differences between TCDD-sensitive or TCDD-resistant models.Sex-dependent sensitivities to TCDD exposure are associated with a set of sex-specific TCDD-responsive genes. In addition, complex interactions between the aryl hydrocarbon and sex hormone receptors may affect the observable differences in sensitivity phenotypes between the sexes. Further work is necessary to better understand the roles of those genes altered by TCDD in a sex-dependent manner, and their association with changes to sex hormones and receptors.
Project description:The process of hematopoiesis, characterized by long-term self-renewal and multi-potent lineage differentiation, has been shown to be regulated in part by the ligand-activated transcription factor known as the aryl hydrocarbon receptor (AHR). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous contaminant and the most potent AHR agonist, also modulates regulation of adult hematopoietic stem and progenitor cell (HSC/HPC) homeostasis. However, the effect of developmental TCDD exposure on early life hematopoiesis has not been fully explored. Given the inhibitory effects of TCDD on hematopoiesis and lymphocyte development, we hypothesized that in utero exposure to TCDD would alter the functional capacity of fetal HSC/HPCs to complete lymphocyte differentiation. To test this hypothesis, we employed a co-culture system designed to facilitate the maturation of progenitor cells to either B or T lymphocytes. Furthermore, we utilized an innovative limiting dilution assay to precisely quantify differences in lymphocyte differentiation between HSC/HPCs obtained from fetuses of dams exposed to 3?g/kg TCDD or control. We found that the AHR is transcribed in yolk sac hematopoietic cells and is transcriptionally active as early as gestational day (GD) 7.5. Furthermore, the number of HSC/HPCs present in the fetal liver on GD 14.5 was significantly increased in fetuses whose mothers were exposed to TCDD throughout pregnancy. Despite this increase in HSC/HPC cell number, B and T lymphocyte differentiation is decreased by approximately 2.5 fold. These findings demonstrate that inappropriate developmental AHR activation in HSC/HPCs adversely impacts lymphocyte differentiation and may have consequences for lymphocyte development in the bone marrow and thymus later in life.
Project description:Environmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation.We performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 mug/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8-16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts.We have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.
Project description:The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.
Project description:We systematically evaluated studies published through May 2014 in which investigators assessed the dose-response relationship between serum levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the occurrence of diabetes mellitus (DM), and we investigated the extent and sources of interstudy heterogeneity. The dose-response relationship between serum TCDD and DM across studies was examined using 2 dependent variables: an exposure level-specific proportion of persons with DM and a corresponding natural log-transformed ratio measure of the association between TCDD and DM. Regression slopes for each dependent variable were obtained for each study and included in a random-effects meta-analysis. Sensitivity analyses were used to assess the influence of inclusion and exclusion decisions, and sources of heterogeneity were explored using meta-regression models and a series of subanalyses. None of the summary estimates in the main models or in the sensitivity analyses indicated a statistically significant association. We found a pronounced dichotomy: a positive dose-response in cross-sectional studies of populations with low-level TCDD exposures (serum concentrations <10 pg/g lipid) and heterogeneous, but on balance null, results for prospective studies of persons with high prediagnosis TCDD body burdens. Considering the discrepancy of results for low current versus high past TCDD levels, the available data do not indicate that increasing TCDD exposure is associated with an increased risk of DM.