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

Chemical sensing in development and function of intestinal lymphocytes.

The immune system of the intestinal tract has the challenging task of recognizing and eliminating intestinal pathogens while maintaining tolerance to dietary and commensal antigens; therefore, it must be able to sense environmental cues within the intestine and mount suitable responses dictated by their pathogenic or nonpathogenic nature. The aryl hydrocarbon receptor (AHR) was originally characterized as a chemical sensor of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [12]. More recently, AHR has emerged as a major chemical sensor expressed in many intestinal immune cells that enables them to distinguish nutritional and microbial cues and is, therefore, important for development, maintenance and function of the intestinal immune system. In this review, we will highlight recent advances in our knowledge of the role of AHR signaling in intestinal innate lymphoid cells (ILC), T cells and B cells.

Actein alleviates 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated cellular dysfunction in osteoblastic MC3T3-E1 cells.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to affect bone metabolism. We evaluated the protective effects of the triterpene glycoside actein from the herb black cohosh against TCDD-induced toxicity in MC3T3-E1 osteoblastic cells. We found that TCDD significantly reduced cell viability and increased apoptosis and autophagy in MC3T3-E1 osteoblastic cells (P < .05). In addition, TCDD treatment resulted in a significant increase in intracellular calcium concentration, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cardiolipin peroxidation, whereas pretreatment with actein significantly mitigated these effects (P < .05). The effects of TCDD on extracellular signal-related kinase (ERK), aryl hydrocarbon receptor, aryl hydrocarbon receptor repressor, and cytochrome P450 1A1 levels in MC3T3-E1 cells were significantly inhibited by actein. The levels of superoxide dismutase, ERK1, and nuclear factor kappa B mRNA were also effectively restored by pretreatment with actein. Furthermore, actein treatment resulted in a significant increase in alkaline phosphatase (ALP) activity and collagen content, as well as in the expression of genes associated with osteoblastic differentiation (ALP, type I collagen, osteoprotegerin, bone sialoprotein, and osterix). This study demonstrates the underlying molecular mechanisms of cytoprotection exerted by actein against TCDD-induced oxidative stress and osteoblast damage.

Indole-3-carbinol, a plant nutrient and AhR-Ligand precursor, supports oral tolerance against OVA and improves peanut allergy symptoms in mice.

In general, dietary antigens are tolerated by the gut associated immune system. Impairment of this so-called oral tolerance is a serious health risk. We have previously shown that activation of the ligand-dependent transcription factor aryl hydrocarbon receptor (AhR) by the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects both oral tolerance and food allergy. In this study, we determine whether a common plant-derived, dietary AhR-ligand modulates oral tolerance as well. We therefore fed mice with indole-3-carbinole (I3C), an AhR ligand that is abundant in cruciferous plants. We show that several I3C metabolites were detectable in the serum after feeding, including the high-affinity ligand 3,3´-diindolylmethane (DIM). I3C feeding robustly induced the AhR-target gene CYP4501A1 in the intestine; I3C feeding also induced the aldh1 gene, whose product catalyzes the formation of retinoic acid (RA), an inducer of regulatory T cells. We then measured parameters indicating oral tolerance and severity of peanut-induced food allergy. In contrast to the tolerance-breaking effect of TCDD, feeding mice with chow containing 2 g/kg I3C lowered the serum anti-ovalbumin IgG1 response in an experimental oral tolerance protocol. Moreover, I3C feeding attenuated symptoms of peanut allergy. In conclusion, the dietary compound I3C can positively influence a vital immune function of the gut.

P69: Dioxin-mediated regulation of expression IL-12 family cytokines in human macrophages as a factor of tumor promotion by TCDD

Background The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic among the dioxin xenobiotics and induces a broad spectrum of biological responses, including immunotoxicity and cancer [1] . Macrophages are very plastic cells and can acquire various functionally distinct phenotypes depending on the physiological context. They are key regulators of the innate immune response and determine the developmental thrust of the adaptive immune response. The action of TCDD on macrophages is unique in that carried out through a variety of pathways, inducing activation of a number of cytokines [2] . Our previous analysis showed that the list of such cytokines was not complete [3] . IL-12 family of cytokines is the key players in the regulation of T cell responses. Little is known of the effects that TCDD has on the expression of IL-12 family member’s genes, such as IL12A, IL12B, IL27 and EBI3, which forms a critically important cytokines IL-12 and IL-27, secreted by human macrophages. In the present work, the effects of TCDD on expression of IL-12 family member’s genes in human macrophages have been investigated. Results To examine the effects of TCDD on the functional characteristics of macrophages, experiments have been conducted on the monocyte-like cell line U937 and primary human macrophages. Obtained data demonstrate functional activity of DREs in IL12A, IL12B and IL4 gene promoters via AhR signal pathway. The mRNA expression dynamics of IL12A, IL12B, IL4, IL27 and EBI3 cytokines genes also evidence the indirect TCDD–mediated modulation of these genes via intrinsic TFs, one of which is ATF3. Conclusion Exposure to TCDD induces a mechanism of two-stage regulation of the expression of some IL-12 family subunits through primary activation of the aryl hydrocarbon receptor by the TCDD-containing complex followed by ATF3-dependent suppression of their expression. This may result in an abrupt reduction of the anti-tumor response mediated by O and NK cells. The observed increase in ATF3 and decrease in IL-12 following exposure to TCDD are consistent with data on the expression of these genes in tumor-associated macrophages (TAMs). Reliance upon available data that demonstrate a decrease in IL-12 alongside an increase in the expression of other cytokines typical of TAMs confirms the hypothesis that TCDD can shift the phenotype of a macrophage towards a tumor-associated direction. This mechanism may be one of the others underlying TCDD-driven tumor promotion. This work was supported by the Russian Foundation for Basic Research (Russia) (No. 12-04-01736 ) and state projects VI.60.1.1 and VI.61.1.2.

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) Increases Bilirubin Formation but Hampers Quantitative Hepatic Conversion of Biliverdin to Bilirubin in Rats with Wild‐Type AH Receptor

In haem degradation, haem oxygenase-1 (HO-1) first cleaves haem to biliverdin, which is reduced to bilirubin by biliverdin IXα reductase (BVR-A). The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin in moderately TCDD-resistant line B (Kuopio) rats. Using line B and two TCDD-sensitive rat strains, the present study set out to probe the dose-response and biochemical mechanisms of this accumulation. At 28 days after exposure to 3-300 μg/kg TCDD in line B rats, already the lowest dose of TCDD tested, 3 μg/kg, affected serum bilirubin conjugates, and after doses ≥100 μg/kg, the liver content of bilirubin, biliverdin and their conjugates (collectively 'bile pigments') as well as HO-1 was elevated. BVR-A activity and serum bile acids were increased only by the doses of 100 and 300 μg/kg TCDD, respectively. Biliverdin conjugates correlated best with biliverdin suggesting it to be their immediate precursor. TCDD (100 μg/kg, 10 days) increased hepatic bilirubin and biliverdin levels also in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. Hepatic bilirubin and bile acids, but not biliverdin, were increased in feed-restricted L-E control rats. In TCDD-sensitive line C (Kuopio) rats, 10 μg/kg of TCDD increased the body-weight-normalized biliary excretion of bilirubin. Altogether, the results suggest that at acutely toxic doses, TCDD induces the formation of bilirubin in rats. However, concurrently, TCDD seems to hamper the quantitative conversion of biliverdin to bilirubin in line B and L-E rats' liver. Biliverdin conjugates are most likely formed as secondary products of biliverdin.

Aryl Hydrocarbon Receptor–Dependent Induction of Liver Fibrosis by Dioxin

The contribution of environmental pollutants to liver fibrosis is an important and poorly explored issue. In vitro studies suggest that the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands induce several genes that are known to be upregulated during liver fibrosis. Our aim was to determine whether exposure to such pollutants can lead to liver fibrosis and to characterize the mechanisms of action. Mice were treated for 2, 14, or 42 days, once a week with 25 µg/kg of TCDD. Gene and protein expression, in vitro and in vivo, as well as liver histology were investigated for each treatment. Treatment of mice with TCDD for 2 weeks modified the hepatic expression of markers of fibrosis such as collagen 1A1 and α-smooth muscle actin. This is not observed in AhR knockout mice. Following 6 weeks of treatment, histological features of murine hepatic fibrosis became apparent. In parallel, the levels of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor α) and of markers of activated fibroblasts(fibroblast-specific protein 1) were found to be upregulated. Interestingly, we also found increased expression of genes of the TGF-β pathway and a concomitant decrease of miR-200a levels. Because the transcription factors of the Snail family were shown to be involved in liver fibrosis, we studied their regulation by TCDD. Two members of the Snail family were increased, whereas their negative targets, the epithelial marker E-cadherin and Claudin 1, were decreased. Further, the expression of mesenchymal markers was increased. Finally, we confirmed that Snai2 is a direct transcriptional target of TCDD in the human hepatocarcinoma cell line, HepG2. The AhR ligand, TCDD, induces hepatic fibrosis by directly regulating profibrotic pathways.

Effects of AhR ligands on the production of immunoglobulins in purified mouse B cells

The aryl hydrocarbon receptor (AhR) has been shown to play important roles in the immune system, and contributions of AhR ligands to the differentiation and functions of Th17/Treg cells have recently been established. However, it has not been fully clarified whether AhR plays roles in B cell differentiation and functions. The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a highly potent AhR agonist, was reported to suppress the production of immunoglobulin M (IgM) in a transformed mouse B cell line. However, TCDD exhibits high toxicity toward cells and has unknown activities except for its action as an AhR agonist. In the present study, we tried to clarify how an endogenously generated AhR agonist affects mouse B cell differentiation and functions in terms of the direct effects on the expression of Ig subclasses in purified mouse B cells stimulated with an anti-CD40 antibody and interleukin-4. The AhR agonist 2-(1'H-indole-3'- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which is derived via tryptophan metabolism, suppressed the expression of not only IgM, but also IgG1 and IgE. ITE was also found to suppress the expression of secreted-type Ig mRNAs and plasma cell-specific genes. These findings indicate that the endogenous AhR agonist suppresses B cell differentiation into Ig-secreting plasma cells.

Combination of 17β-estradiol with the environmental pollutant TCDD is involved in pathogenesis of endometriosis via up-regulating the chemokine I-309–CCR8

To explore the effects of the combined E(2) with the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on CCR8-I-309 expression by the endometriotic lesion-associated cells in the pathogenesis of endometriosis. Prospective laboratory study. University hospital. Chinese women with endometriosis. The endometriotic tissue and matched eutopic endometrium were collected. Endometrial stromal cells (ESCs), HPMC, and U937 cells were treated with 17beta-E(2) or TCDD. The ESCs were stimulated with I-309. The expression of CCR8 in tissues was analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. The effect of I-309 on integrin beta1 and alphavbeta3 expression intensity was analyzed by flow cytometry, and the chemotactic activity of I-309 on the ESC was explored by chemotactic assay. Concentration of I-309 in the culture supernatant was quantified by enzyme-linked immunosorbent assay. CCR8 was overexpressed in the endometriotic tissue. I-309 promoted the expression of integrin beta1. Estradiol and TCDD up-regulated CCR8 expression by ESCs. Estradiol magnified the stimulatory effect of TCDD on I-309 secretion by U937. The interaction of HPMC and U937 cells promoted I-309 secretion. These findings imply that the combination of 17beta-E(2) with the environmental pollutant TCDD is involved in the pathogenesis of endometriosis via up-regulating the chemokine CCR8-I-309.

Comparative uterine gene expression analysis after dioxin and estradiol administration

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many organisms. TCDD exposure is known to be associated with abnormal development, hepatotoxicity and endocrine effects. It has also been reported to have antiestrogenic activity in addition to estrogenic activity. In order to clarify the effects of TCDD in the uterus, we evaluated the patterns of gene expression after TCDD and estradiol administration. Of the 10 000 arrayed genes, only a few were affected by both estradiol and TCDD. Although the subset of genes that responded to estrogen was also activated by TCDD, the response to TCDD was more limited than that observed in response to estradiol. Therefore, according to our analysis of gene expression patterns, TCDD had partial and weak estrogenic activity in the uterus.

2,3,7,8-Tetrachlorodibenzo- p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all- trans-retinoic acid (atRA) formation. Male Sprague–Dawley rats were exposed to a single oral dose of TCDD in a combined dose–response and time–course study, with doses ranging from 0.1 to 100 μg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin- O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time–course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.

Conditional expression of a constitutively active aryl hydrocarbon receptor in MCF-7 human breast cancer cells

In addition to inducing transcription of a battery of target genes encoding drug-metabolizing enzymes, the environmental pollutant 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is known to induce antiestrogenic responses. However, the mechanisms underlying such complex biologic responses affecting growth and differentiation remain unclear. In the present study we have investigated biological effects of a constitutively active mutant of the aryl hydrocarbon (Ah) receptor (CA-AhR), in particular whether it modulates estrogen receptor function in human MCF-7 breast cancer cells. To this end, the CA-AhR protein was conditionally expressed using the tet repressor. Expression of CA-AhR resulted in constitutive formation of a DNA-binding AhR–aryl hydrocarbon receptor nuclear translocator heterodimeric complex and enhanced expression of the Ah receptor target gene CYP1A1 in the absence of TCDD. Moreover, expression of CA-AhR inhibited estrogen-dependent cathepsin D expression and growth of these cells. Thus, the present model system conditionally expressing the CA-AhR protein provides a novel tool for the investigation of AhR-mediated signaling pathways.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Affects Keratin 1 and Keratin 17 Gene Expression and Differentially Induces Keratinization in Hairless Mouse Skin

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes chloracne in humans by mechanisms that are as yet poorly understood. Because TCDD is known to affect keratinocyte differentiation in vitro, we have studied TCDD-dependent morphologic changes and the expression of murine keratin 1 (MK1; differentiation associated) and keratin 17 (MK17; presumably hyperproliferation associated) in HRS/J hr/hr hairless mouse skin. TCDD (0.2 microg in acetone) applied topically to the dorsal skin caused epidermal acanthosis and hyperkeratosis of the dermal cysts as well as an involution of the utricles and the sebaceous glands. By means of in situ hybridization with digoxigenin-labeled riboprobes of sections from untreated and vehicle (control)-treated skin, we localized MK1 mRNA to the epidermal spinous cell compartment. MK17 transcripts were detected only in the derivatives of the hair follicle-utricle epithelium and dermal cysts. No spatial overlap was observed between MK1 and MK17 expression. After TCDD application, MK17 was newly expressed in the upper spinous cell layers of the interfollicular epidermis, although it was suppressed in the involuting utricles. In contrast, MK1 expression in the interfollicular epidermis was not affected by TCDD. Furthermore, MK1 expression was induced in the epithelium of the utricle remnants and in some dermal cysts. These data suggest that increased keratinization of the part of the follicular epithelium corresponding to the dermal cyst epithelium of hairless mice most probably explains the pathogenesis of TCDD-induced chloracne. The results demonstrate, furthermore, that TCDD can differentially affect keratinocyte differentiation in vivo as well as in vitro.

Increased copper concentrations in rat tissues after acute intoxication with 2,3,7,8-tetrachlorodibenzo-p-dioxin

Recently, acutely toxic doses of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been reported to affect the hepatic distribution of essential metals in the rat. However, the reduced food intake by TCDD was not taken into account. Therefore, metal concentrations were determined in different rat tissues at the end of a toxicity study with TCDD in which a pair-fed control group was introduced. Male Sprague-Dawley rats received a single i.p. injection of corn oil/acetone with or without TCDD at 125 micrograms/kg. Controls and TCDD-treated rats were fed ad libitum; additionally, pair-fed controls received the amount of food consumed by their TCDD-treated partners 1 day before. Twenty-one days after dosing rats were killed and samples of liver, kidney and jejunum were taken for the analysis of Ca, Cu, Fe, Mg, Mn, and Zn. After acid digestion of the tissues metals were determined by atomic emission spectrometry (AES). The most outstanding effect of TCDD treatment was an increase of the copper levels in the kidney (4-fold, versus pair-fed controls) and in the liver (greater than 2-fold, versus pair-fed controls). Other metals were mainly affected by the reduced food intake only. Since Cu represents a trace metal the homeostasis of which depends on its biliary excretion and since TCDD is known to impair biliary flow and excretion, an impaired biliary excretion of Cu by TCDD is suggested as the causal mechanism.