Molecular Targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the Zebrafish Ovary: Insights into TCDD-induced Endocrine Disruption and Reproductive Toxicity

TCDD is a reproductive toxicant and endocrine disruptor, yet the mechanisms by which it causes these reproductive alterations are not fully understood. In order to provide additional insight into the molecular mechanisms that underlie TCDD\u27s reproductive toxicity, we assessed TCDD-induced transcriptional changes in the ovary as they relate to previously described impacts on serum estradiol concentrations and altered follicular development in zebrafish. In silico computational approaches were used to correlate candidate regulatory motifs with observed changes in gene expression. Our data suggest that TCDD inhibits follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis, and that interference of estrogen-regulated signal transduction may also contribute to TCDD\u27s impacts on follicular development. TCDD may also alter ovarian function by disrupting various signaling pathways such as glucose and lipid metabolism, and regulation of transcription. Furthermore, events downstream from initial TCDD molecular-targets likely contribute to ovarian toxicity following chronic exposure to TCDD. Data presented here provide further insight into the mechanisms by which TCDD disrupts follicular development and reproduction in fish, and can be used to formulate new hypotheses regarding previously documented ovarian toxicity

Prenatal exposure to TCDD and atopic conditions in the Seveso second generation: a prospective cohort study.

Background2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic environmental contaminant that can bioaccumulate in humans, cross the placenta, and cause immunological effects in children, including altering their risk of developing allergies. On July 10, 1976, a chemical explosion in Seveso, Italy, exposed nearby residents to a high amount of TCDD. In 1996, the Seveso Women's Health Study (SWHS) was established to study the effects of TCDD on women's health. Using data from the Seveso Second Generation Health Study, we aim to examine the effect of prenatal exposure to TCDD on the risk of atopic conditions in SWHS children born after the explosion.MethodsIndividual-level TCDD was measured in maternal serum collected soon after the accident. In 2014, we initiated the Seveso Second Generation Health Study to follow-up the children of the SWHS cohort who were born after the explosion or who were exposed in utero to TCDD. We enrolled 677 children, and cases of atopic conditions, including eczema, asthma, and hay fever, were identified by self-report during personal interviews with the mothers and children. Log-binomial and Poisson regressions were used to determine the association between prenatal TCDD and atopic conditions.ResultsA 10-fold increase in 1976 maternal serum TCDD (log10TCDD) was not significantly associated with asthma (adjusted relative risk (RR) = 0.93; 95% CI: 0.61, 1.40) or hay fever (adjusted RR = 0.99; 95% CI: 0.76, 1.27), but was significantly inversely associated with eczema (adjusted RR = 0.63; 95% CI: 0.40, 0.99). Maternal TCDD estimated at pregnancy was not significantly associated with eczema, asthma, or hay fever. There was no strong evidence of effect modification by child sex.ConclusionsOur results suggest that maternal serum TCDD near the time of explosion is associated with lower risk of eczema, which supports other evidence pointing to the dysregulated immune effects of TCDD

Proteomic analysis of the rat ovary following chronic low-dose exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitously distributed endocrine-disrupting chemical and reproductive toxicant. In order to elucidate low-dose TCDD-mediated effects on reproductive or endocrine functions, female Sprague-Dawley rats were orally administered various concentrations (20, 50, or 125 ng/kg once weekly) TCDD for 29 wk. A proteomic analysis of the ovaries by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry showed distinct changes in the levels of several proteins that are relevant markers of TCDD toxicity. Serum estradiol (E2) levels of TCDD-treated animals were markedly lower than control. There were no significant differences in bone mineral density (BMD) of femurs. The body weight of the 125-ng/kg TCDD group was significantly decreased relative to control and there was also a significant reduction in absolute and relative ovarian weights. Expressions of selenium binding protein 2, glutathione S-transferase mu type 3, Lrpap1 protein, NADPH, and peptidylprolyl isomerase D were upregulated, while prohibitin and N-ethylmaleimide-sensitive factor expression levels were downregulated. Data provide further insight into the mechanisms by which TCDD disrupts ovarian function by indicating which differential protein expressions following low-dose TCDD exposure

2,3,7,8‑Tetrachlorodibenzo‑p‑dioxin suppresses the growth of human liver cancer HepG2 cells in vitro: Involvement of cell signaling factors.

The aryl hydrocarbon receptor (AHR) is transcriptionally active in the form of a heterodimer with the AHR nuclear translocator, which then binds to the xenobiotic responsive element. AHR was originally discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8‑tetrachlorodibenzo‑p‑dioxin (TCDD). In this study, we investigated whether TCDD regulates the growth of human liver cancer HepG2 cells in vitro. TCDD (0.1‑100 nM) was found to exert suppressive effects on the colony formation and proliferation of HepG2 cells, and stimulatory effects on the death of HepG2 cells when the cells reached subconfluence. The effects of TCDD on the HepG2 cells were abolished by culture with CH223191, an inhibitor of AHR signaling. The effects of TCDD were dependent on the concentration of serum, which contains various signaling factors. The effects of TCDD were not potentiated by culture with tumor necrosis factor‑α, which activates the signaling of nuclear factor‑κB (NF‑κB). The results of western blot analysis revealed that TCDD increased the protein levels of p53, Rb, p21, and regucalcin, which are suppressors of the growth of tumor cells. Moreover, TCDD enhanced the NF‑κB p65, β‑catenin, signal transducer and activator of transcription 3 (STAT3), Ras and Akt levels. Thus, the findings of this study indicate that TCDD may suppress liver cancer cell growth through various signaling pathways, mediated by AHR and its‑related co‑factors. Of note, the effects of TCDD were found to be potentiated by gemcitabine, which induces nuclear DNA damage in cancer cells, suggesting that their combined use may have potential as a suppressor of tumor cell growth

Detection of the TCDD binding-fingerprint within the Ah receptor ligand binding domain by structurally driven mutagenesis and functional analysis

The aryl hydrocarbon receptor (AhR) is a ligand-dependent, basic helix-loop-helix Per-Arnt-Sim (PAS)-containing transcription factor that can bind and be activated by structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Our previous three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain allowed identification of the binding site and its experimental validation. We have extended this analysis by conducting comparative structural modeling studies of the ligand binding domains of six additional highaffinity mammalian AhRs. These results, coupled with site-directed mutagenesis and AhR functional analysis, have allowed detection of the "TCDD binding-fingerprint" of conserved residues within the ligand binding cavity necessary for high-affinity TCDD binding and TCDD-dependent AhR transformation DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD with both wild-type and mutant mAhRs. Taken together, our results dramatically improve our understanding of the molecular determinants of TCDD binding and provide a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure. © 2009 American Chemical Society

Serum dioxin concentrations and endometriosis: a cohort study in Seveso, Italy.

Dioxin, a ubiquitous contaminant of industrial combustion processes including medical waste incineration, has been implicated in the etiology of endometriosis in animals. We sought to determine whether dioxin exposure is associated with endometriosis in humans. We conducted a population-based historical cohort study 20 years after the 1976 factory explosion in Seveso, Italy, which resulted in the highest known population exposure to 2,3,7,8-tetrachlorodibenzo-(italic)p(/italic)-dioxin (TCDD). Participants were 601 female residents of the Seveso area who were (3/4) 30 years old in 1976 and had adequate stored sera. Endometriosis disease status was defined by pelvic surgery, current transvaginal ultrasound, pelvic examination, and interview (for history of infertility and pelvic pain). "Cases" were women who had surgically confirmed disease or an ultrasound consistent with endometriosis. "Nondiseased" women had surgery with no evidence of endometriosis or no signs or symptoms. Other women had uncertain status. To assess TCDD exposure, individual levels of TCDD were measured in stored sera collected soon after the accident. We identified 19 women with endometriosis and 277 nondiseased women. The relative risk ratios (RRRs) for women with serum TCDD levels of 20.1-100 ppt and >100 ppt were 1.2 [90% confidence interval (CI) = 0.3-4.5] and 2.1 (90% CI = 0.5-8.0), respectively, relative to women with TCDD levels (3/4) 20 ppt. Tests for trend using the above exposure categories and continuous log TCDD were nonsignificant. In conclusion, we report a doubled, nonsignificant risk for endometriosis among women with serum TCDD levels of 100 ppt or higher, but no clear dose response. Unavoidable disease misclassification in a population-based study may have led to an underestimate of the true risk of endometriosis

Comparative analysis of homology models of the Ah receptor ligand binding domain: Verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness. © 2013 American Chemical Society

Altering HIF-1α through 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure affects coronary vessel development.

Differential tissue hypoxia drives normal cardiogenic events including coronary vessel development. This requirement renders cardiogenic processes potentially susceptible to teratogens that activate a transcriptional pathway that intersects with the hypoxia-inducible factor (HIF-1) pathway. The potent toxin 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause cardiovascular defects by way of reduced myocardial hypoxia, inhibition of angiogenic stimuli, and alterations in responsiveness of endothelial cells to those stimuli. Our working hypothesis is that HIF-1 levels and thus HIF-1 signaling in the developing myocardium will be reduced by TCDD treatment in vivo during a critical stage and in particularly sensitive sites during heart morphogenesis. This inadequate HIF-1 signaling will subsequently result in outflow tract (OFT) and coronary vasculature defects. Our current data using the chicken embryo model showed a marked decrease in the intensity of immunostaining for HIF-1α nuclear expression in the OFT myocardium of TCDD-treated embryos. This area at the base of the OFT is particularly hypoxic during normal development; where endothelial cells initially form a concentrated anastomosing network known as the peritruncal ring; and where the left and right coronary arteries eventually connect to the aortic lumen. Consistent with this finding, anomalies of the proximal coronaries were detected after TCDD treatment and HIF-1α protein levels decreased in a TCDD dose-dependent manner