To The Environmental Contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin Research Articles

AKT/mTOR-mediated autophagic signaling is associated with TCDD-induced cleft palate

In utero exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can contribute to high rates of cleft palate (CP) formation, but the mechanistic basis for these effects remains uncertain. Here, multi-omics-based metabolomic and transcriptomic analyses were employed to characterize the etiological basis for TCDD-induced CP on gestational day 14.5 (GD14.5). These analyses revealed that TCDD-induced CP formation is associated with calcium, MAPK, PI3K-Akt, and mTOR pathway signaling. PI3K-Akt and mTOR signaling activity is closely linked with the maintenance of cellular proliferation and survival. Moreover, mTOR-mediated regulation of autophagic activity is essential for ensuring an appropriate balance between metabolic activity and growth. Murine embryonic palatal mesenchymal (MEPM) cell proliferation was thus characterized, autophagic activity in these cells was evaluated through electron microscopy and western immunoblotting was used to compare the levels of autophagy- and AKT/mTOR-related protein between the control and TCDD groups on GD14.5. These analyses indicated that MEPM cell proliferative and autophagic activity was inhibited in response to TCDD exposure with the concomitant activation of AKT/mTOR signaling, in line with the multi-omics data. Together, these findings suggested that following TCDD exposure, the activation of AKT/mTOR-related autophagic signaling may play a role in the loss of appropriate palatal cell homeostasis, culminating in the incidence of CP.

Upregulated LncRNA-Meg3 modulates the proliferation and survival of MEPM cells via interacting with Smad signaling in TCDD-induced cleft palate

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero can result in high rates of cleft palate (CP) formation, yet the underlying mechanisms remain to be characterized. In vivo, the lncRNA Meg3 was upregulated following TCDD treatment in CP-associated murine embryonic palatal tissue, with concomitant changes in proliferative and apoptotic activity in these murine embryonic palatal mesenchymal (MEPM) cells. Meg3 can modulate the TGF-β/Smad to control the proliferation, survival, and differentiation of cells. Accordingly, TCCD and TGF-β1 were herein used to treat MEPM cells in vitro, revealing that while TCDD exposure altered the proliferative activity and apoptotic death of these cells, exogenous TGF-β1 exposure antagonized these effects via TGF-β/Smad signaling. TCDD promoted Meg3 upregulation, whereas TGF-β1 suppressed TCDD-driven upregulation of this lncRNA. Meg3 was additionally determined to directly interact with Smad2, with significant Meg3 enrichment in Smad2-immunoprecipitates following TCDD treatment. When Meg3 was silenced, the impact of TCDD on Smad signaling, proliferative activity, and apoptosis were ablated, while the effects of exogenous TGF-β1 were unchanged. This supports a model wherein Meg3 is upregulated in TCDD-exposed palatal tissue whereupon it can interact with Smad2 to suppress Smad-dependent signaling, thus controlling MEPM cell proliferation and apoptosis, contributing to TCDD-induced CP, which provides a theoretical support for the precautions of cleft palate induced by TCDD.

High-Resolution Genome-wide Mapping of AHR and ARNT Binding Sites by ChIP-Seq

The aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) activated complex regulates genes in response to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has also emerged as a potential therapeutic target for the treatment of human diseases and different cancers, including breast cancer. To better understand AHR and ARNT signaling in breast cancer cells, we used chromatin immunoprecipitation linked to high-throughput sequencing to identify AHR- and ARNT-binding sites across the genome in TCDD-treated MCF-7 cells. We identified 2594 AHR-bound, 1352 ARNT-bound, and 882 AHR/ARNT cobound regions. No significant differences in the genomic distribution of AHR and ARNT were observed. Approximately 60% of the cobound regions contained at least one core an aryl hydrocarbon response element (AHRE), 5'-GCGTG-3'. AHR/ARNT peak density was the highest within 1 kb of transcription start sites (TSS); however, a number of AHR/ARNT cobound regions were located as far as 100 kb from TSS. De novo motif discovery identified a symmetrical variation of the AHRE (5'-GTGCGTG-3'), as well as FOXA1 and SP1 binding motifs. Microarray analysis identified 104 TCDD-responsive genes where 98 genes were upregulated by TCDD. Of the 104 regulated genes, 69 (66.3%) were associated with an AHR- or ARNT-bound region within 100 kb of their TSS. Overall our study identified AHR/ARNT cobound regions across the genome, revealed the importance but not absolute requirement for an AHRE in AHR/ARNT interactions with DNA, and identified a modified AHRE motif, thereby increasing our understanding of AHR/ARNT signaling pathway.

In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Amphiregulin Gene Expression in the Developing Mouse Ureter

Exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis in developing mice, the etiology of which involves hyperplasia within the ureteric luminal epithelium. Dysregulation of epidermal growth factor receptor (EGFR), EGF, and transforming growth factor-alpha expression has been implicated as playing a role in TCDD-induced hydronephrosis. In this study, changes in the expression of genes encoding the EGFR and its cognate ligands in response to TCDD were evaluated within the developing ureter. C57BL/6 dams were injected ip with 30 mug/kg TCDD on gestational day (GD) 13 or 16 and fetal tissues removed on GD 17. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator messenger RNA (mRNA) were expressed in control and treated fetal tissues at GD 14 and 17. Prototypical AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 were upregulated in TCDD-exposed fetal tissues, demonstrating AHR transcriptional activity at these developmental stages. Amphiregulin (AREG) and epiregulin, ligands for the EGFR, were induced at the transcriptional level in ureters of fetuses exposed to TCDD for 24 h. AREG mRNA was also induced by TCDD dose- and time-dependently in the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1), mimicking the induction patterns of CYP1A1 mRNA. Other AHR ligands also induced AREG mRNA in Hepa-1 cells. Furthermore, variant Hepa-1 cells (TAOBP(r)c1 cells) virtually deficient in the AHR failed to display an increase in AREG mRNA in response to TCDD. Taken together, these data suggest that the AHR cross talks with the EGFR signaling pathway by directly inducing the expression of growth factors that are important for EGFR signaling in the developing mouse ureter.

Interaction between the Aryl Hydrocarbon Receptor and Retinoic Acid Pathways Increases Matrix Metalloproteinase-1 Expression in Keratinocytes

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of pathological lesions in humans via activation of the aryl hydrocarbon receptor (AhR) pathway. It has become apparent that this pathway interacts with a variety of signaling pathways that are believed to be involved in mediating TCDD/AhR biological effects. Our hypothesis is that TCDD mediates these pathological lesions by directly altering the expression of genes involved in matrix deposition and remodeling and that the retinoic acid signaling pathway is involved in modulating TCDD-induced effects. Therefore, we examined the effect of TCDD and all-trans retinoic acid (atRA) on the expression of matrix metalloproteinase-1 (MMP-1, interstitial collagenase), one of the proteolytic enzymes that degrade type I collagen, in normal human keratinocytes. The data show that TCDD exposure results in increased MMP-1 expression in keratinocytes that is further enhanced by co-treatment with all-trans retinoic acid. TCDD-induced expression of MMP-1 appears to be mediated through two AP-1 elements in the proximal promoter of the MMP-1 gene. However, retinoic acid-mediated induction of keratinocyte MMP-1 is a result of both promoter activation and increased mRNA stability. These findings are the first to demonstrate TCDD-induced expression of MMP-1 and to demonstrate interactions between the TCDD/AhR and retinoic acid pathways on MMP-1 expression.

Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression

One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5×7.5 cm) covering a surface area of 2.25 cm 2. We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.

The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11.10 transgenic CD4(+) T cells in adoptively transferred mice.

Exposure to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses the generation of T cell-dependent immunity, both humoral and cell-mediated. However, the mechanism of TCDD-induced immune suppression remains to be defined. We hypothesized that exposure to TCDD suppresses the activation of naive CD4(+) T cells and prevents their expansion and differentiation into effector T-helper cells capable of driving T cell-dependent immune responses. To test this hypothesis, we adoptively-transferred DO11.10 OVA-specific T-cell receptor (TCR) transgenic T cells into syngeneic recipients and used a TCR-specific monoclonal antibody to track the in vivo activation of naive CD4(+) T lymphocytes following exposure to OVA. The production of OVA-specific antibodies was suppressed in a dose-dependent manner in adoptively transferred mice that had been exposed to TCDD. Although TCDD exposure had little effect on the expansion or activation of the adoptively transferred, OVA-specific CD4(+) T cells, these cells disappeared from the spleen more rapidly in TCDD-treated mice and produced significantly decreased levels of the T cell-derived cytokines IL-2 and IL-10. There was also a trend towards reduced IFN-gamma and IL-4 production following in vitro re-stimulation. These data suggest that TCDD may interfere with the survival and/or differentiation of OVA-specific T-helper cells. These results demonstrate for the first time the potential of the DO11.10 adoptive transfer system to directly assess immunotoxic effects of xenobiotics on antigen-specific CD4(+) T cells in vivo.

LEUKOCYTE HYPOCELLULARITY IN THE SPLEEN AND PRONEPHROS OF TILAPIA (OREOCHROMIS NILOTICUS) EXPOSED TO 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) MAY RESULT FROM ANTIPROLIFERATIVE EFFECTS AND ENHANCED APOPTOSIS

Tilapia (Oreochromis niloticus) were subacutely exposed to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by intraperitoneal injection of 1 or 5 mug/kg/day for five consecutive days. Cellularity and morphology of the spleen and pronephros were evaluated two days following dosing. Total cell counts in both the spleen and pronephros were significantly reduced by the 5-mug/kg but not the 1-mug/kg dose of TCDD. Cellular depletion was evident histologically at the higher dose level, particularly within lymphoid regions of the fish spleen and pronephros. Increased numbers of apoptotic cells were observed in the histologic preparations. Light microscopic analysis of cytospin samples from the pronephros of chemical-treated fish verified the increased incidence of cells displaying features typical of apoptosis (condensed nuclei and cytoplasmic fragmentation). Subsequent flow cytom etric evaluation using the DNA-binding dye, 7-aminoactinomycin D (7-AAD), demonstrated a population of apopto...

A Direct Interaction between the Aryl Hydrocarbon Receptor and Retinoblastoma Protein: LINKING DIOXIN SIGNALING TO THE CELL CYCLE

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor in eukaryotic cells that alters gene expression in response to the environmental contaminant 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD). In 5L hepatoma cells, TCDD induces a G1 cell cycle arrest through a mechanism that involves the AhR. The retinoblastoma tumor suppressor protein (pRb) controls cell cycle progression through G1 in addition to promoting differentiation. We examined whether the human AhR or its dimerization partner, the AhR nuclear translocator, interacts with pRb as a basis of the TCDD-induced cell cycle arrest. In vivo and in vitro assays reveal a direct interaction between pRb and the AhR but not the AhR nuclear translocator protein. Binding between the AhR and pRb occurs through two distinct regions in the AhR. A high affinity site lies within the N-terminal 364 amino acids of the AhR, whereas a lower affinity binding region colocalizes with the glutamine-rich transactivation domain of the receptor. AhR ligand binding is not required for the pRb interaction per se, although immunoprecipitation experiments in 5L cells reveal that pRb associates preferentially with the liganded AhR, consistent with a requirement for ligand-induced nuclear translocation. These observations provide a mechanistic insight into AhR-mediated cell cycle arrest and a new perspective on TCDD-induced toxicity.

Different In Vitro Systems Affect CYPIA1 Activity in Response to 2,3,7,8-Tetrachlorodibenzo- p-dioxin

The induction of cytochrome P450IA1 (CYP1A1) activity in human hepatoma cell lines has been used as a model response for exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). CYP1A1 induction is mediated by the intracellular Ah receptor. We have designed a cell culture analogue system to evaluate the effect of low dose continuous exposures to TCDD such that the responses can be compared with traditional static in vitro exposures. The two-compartment model is designed to mimic aspects of time-dependent dose exposure in humans and consists of a ‘liver’ compartment with HepG2 cells and an ‘other tissues’ compartment. Exposure of microcarrier-attached HepG2 cells in spinner flask to a single dose of TCDD resulted in an EC 50 for ethoxyresorufin- o-deethylase (EROD) activity induction of 1.49 n m. Using a one-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of 0.69 n m TCDD. Finally, using a two-compartment reactor with continuous TCDD dosing resulted in an EC 50 for EROD induction of about 0.05 n m. Parallel studies using 3H-TCDD were performed to determine the amount of cell associated 3H-TCDD and subsequently to estimate the amount of bound Ah receptor. CYPIA1 mediated EROD activities in both cell lines correlated with the estimated amount of bound Ah receptor. To illustrate how these systems might alter estimates of risk, the data were evaluated to estimate the TCDD level which would increase CYPIA1 mediated EROD activity to 0.01% of the maximal response. The two-compartment reactor data yielded an estimate of allowable TCDD exposure of 1×10 −5 n m for an acceptable ‘risk’ level of 0.01%. In contrast, values were estimated as 4×10 −4 n m from the one compartment reactor and 4×10 −3 n m from spinner flasks. This work demonstrates the importance of design of the in vitro system on risk assessment.

Down-regulation of Major Histocompatibility ComplexQ1b Gene Expression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

Abstract We analyzed mouse hepatoma cells using differential display to discover new genes that respond to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We identified a class I major histocompatibility complex (MHC) gene, which we designated as MHC Q1 b, whose expression decreases in the presence of TCDD. TCDD-induced down-regulation of MHCQ1 b requires both the aromatic hydrocarbon receptor and the aromatic hydrocarbon receptor nuclear translocator, transcription factors that up-regulate other genes in response to TCDD. Down-regulation of MHC Q1 b by TCDD appears to involve both transcriptional and post-transcriptional regulatory events; the post-transcriptional destabilization of MHCQ1 b mRNA is probably a secondary response to TCDD. Our findings reveal new mechanistic aspects of gene regulation by TCDD. In addition, our observations suggest a mechanism that might account for some of TCDD’s immunotoxic effects.

SUBACUTE EXPOSURE TO 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN GERIATRIC MICE: EFFECTS IN THYMIC, SPLENIC, AND BONE MARROW HEMATOPOIETIC CELLS

Exposure of geriatric m ice (21- 1 m o) to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produced alterations in thymus, spleen, and bone m arrow hematopoietic cell populations. Female C57BL/ 6 mice were dosed (via intraperitoneal injection) with 0, 5.0, or 10.0 ug/ kg TCDD in corn oil for 5 consecutive days. Evaluation of imm une endpoints took place on the second day following cessation of dosing. A limited but significant reduction in bone marrow cellularity was produced by exposure to TCDD at 10.0 ug/ kg, in the presence of unaltered percentages of cells expressing CD45, CD45R, and Mac-1 antigens. A lim ited increase in splenic cellularity was also present at the higher dose level of TCDD, where trends toward reduced percent++ ages of CD45R B cells and Thy 1.2 T cells were observed but did not reach significance. Pronounced, dose-dependent thymic hypocellularity was evident at both levels of chemical exposure in the aged m ice, and was accom panied by+ + decreased percentages of CD4 CD8 (double positive, DP) cells and concomitant--- + increased percentages of CD4 CD8 (double negative, DN) and CD4 CD8 (single positive, SP) cells. The total numbers of thymocytes in all four phenotypes defined by CD4 and CD8 antigens decreased significantly in treated animals, an apparent reflection of the chem ical-induced hypocellularity. These results indicate that, sim ilar to perinatal and young adult m odels, the geriatric murine thym us may be a highly sensitive target of TCDD exposure.

Transcriptional Activation by the Mouse Ah Receptor.: INTERPLAY BETWEEN MULTIPLE STIMULATORY AND INHIBITORY FUNCTIONS

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates cellular responses to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We cloned AhR cDNA from C57BL/6 mouse liver and verified by transfection that it encodes a functional protein. Analyses of deletion mutants indicate that the carboxyl half of AhR contains several types of transactivation domain, which function independently of domains that mediate TCDD recognition, DNA binding, and heterodimerization with the Ah receptor nuclear translocator (Arnt) protein. The transactivation domains function independently of each other, display different levels of activity, and act synergistically when linked. In addition, AhR contains an 82-amino acid domain that inhibits transactivation. The inhibitory domain displays specificity, in that it blocks the transactivating functions of AhR and Arnt, but not that of the herpes simplex protein VP16. The inhibitory activity depends upon the cell type in which AhR is expressed, implying that a cell-specific protein mediates the effect.

Phenotypic Analysis of Spleen, Thymus, and Peripheral Blood Cells in Aged C57B1/6 Mice Following Long-Term Exposure to 2,3,7,8-Tetrachlorodibenzo- p-Dioxin

Phenotypic Analysis of Spleen, Thymus, and Peripheral Blood Cells in Aged C57B1/6 Mice Following Long-Term Exposure to 2,3,7,8-Tetrachlorodibenzo- p-Dioxin. Oughton, J. A., Pereira, C. B., Dekrey, G. K., Collier, J. M., Frank, A. A., and Kerkvliet N. I. (1995). Fundam. Appl. Toxicol. 25, 60-69. A mouse model was used to identify potential biomarkers of exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Female C57B1/6 mice were treated weekly with 0.2 μg TCDD/kg body weight or vehicle for 14-15 months. Phenotypic analysis by flow cytometry identified the major cell subpopulations in the spleen, thymus, and peripheral blood as defined by the expression of CD4, CD8, B220, and Mac-1 molecules. These subpopulations were further characterized for the expression of I-A, Pgp-1, CD45RB, and/or T cell receptor antigens (CD3, αβ, γδ). A group of young (4 months old) mice was evaluated concurrently to document immunophenotype alterations associated with aging. Results showed several age-related changes in phenotype distribution in the spleen and blood, but not in the thymus, despite significant age-dependent thymic involution. The age-dependent changes in splenic phenotypes included a decreased frequency of CD4 + cells and a major shift in tile frequency distribution from naive T cells to effector and memory T cells as defined by Pgp-1 and CD45RB expression. These phenotypic changes in the spleen due to aging correlated with similar changes in the blood, providing preliminary support for the use of spleen cells as surrogates for blood in the development of biomarkers of immunotoxicity. Long-term exposure to a total cumulative dose 12-13 μg TCDD/kg body weight resulted in no overt toxicity, a 16-fold elevation of hepatic ethoxyresorufin- O-deethylase activity, and residue levels of 1.27 ± 0.16 ng TCDD/g abdominal fat. In comparison to the effects of aging, TCDD treatment produced relatively subtle changes in immunophenotypes. In the TCDD-treated thymus, the proportion of CD4 -CD8 - cells was increased as was the proportion of γδ + thymocytes. These effects were very small but of interest in that similar thymic effects have been previously reported following prenatal exposure to TCDD. In the spleen, TCDD exposure did not alter the frequency of CD4 + or CD8 + T cells, B cells, or macrophages but significantly altered functionally discrete subpopulations within the T cell compartment. The most definitive change in TCDD-treated mice was a decrease in the frequency of memory T helper cells, defined as CD4 + Pgp-1 hiCD45RB lo, with a concomitant increase in the proportion of naive T helper cells identified as CD4 +Pgp-1 loCD45RB hi. These changes are consistent with the known immunosuppressive activity of TCDD. Thus, these results identify Pgp-1 and CD45RB as potential biomarkers of TCDD immunotoxicity.

Structure of the 5′ Flanking Region of Class 3 Aldehyde Dehydrogenase in the Rat

Class 3 aldehyde dehydrogenase (ALDH-3) is induced by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and during chemical carcinogenesis. These inductions as well as the basal expression of ALDH-3 vary significantly in different organs. In order to identify DNA elements controlling ALDH-3 expression, we have cloned and analyzed approximately 5.5 kb of the 5′ flanking region of the ALDH-3 gene. Deletion analysis showed that the 5′ flanking region contains at least three functional domains: a strong promoter proximal to the transcription start site, inhibitory regions upstream of the promoter, and TCDD-responsive enhancers. The TCDD-responsive enhancers in the ALDH-3 gene were functionally similar to xenobiotic responsive elements in the cytochrome P450IA1 gene. These results indicate that transcription of the ALDH-3 gene is controlled by cooperation of at least three functional domains.