The aromatic hydrocarbon receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and variable levels of induced aryl hydrocarbon hydroxylase activity in clones of mouse hepatoma cells

Induction of aryl hydrocarbon hydroxylase (AHH) activity was studied in clones and subclones of mouse hepatoma (Hepa-lcl) cells. When maximally induced, one clone had significantly lower (p less than 0.005), two had approximately the same, and two had significantly higher (p less than 0.005) levels of AHH activity compared with Hepa-lcl. The maximal level of induced activity, relative to the parent population, in two clones chosen for further analysis was 0.14 +/- 0.09 for clone 1 (Hs-1) and 0.94 +/- 0.28 for clone 9 (Hs-9). These relative levels were stable over a period of 10 months and were similar when activity was induced either with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benz[a]anthracene. Subclones of Hepa-lcl cells, derived from the Hs-9 clone, also demonstrated variation in induced AHH activity. When maximally induced with TCDD, six subclones had significantly lower AHH activity (p less than 0.005), two had approximately the same, and one had significantly higher levels (p less than 0.005) compared with the progenitor Hs-9 population. Comparative analysis of Ah receptor characteristics in two unselected clones of Hepa-lcl with significantly different levels of AHH activity demonstrated that there was no apparent correlation between relative level of induced AHH activity and (i) total quantity of Ah receptor (cytosol and nuclear), (ii) receptor affinity for TCDD and number of receptor sites in each cell, (iii) subcellular distribution of [3H]TCDD, or (iv) specificity and saturable nature of binding. Coordinate measurement of the concentration of nuclear receptor and absolute induced AHH activity in Hepa-lcl and its clones had a positive correlation (r = 0.79).     

Ovarian endocrine disruption underlies premature reproductive senescence following environmentally relevant chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin

The aryl hydrocarbon receptor (AHR) mediates the effects of many endocrine disruptors and contributes to the loss of fertility in polluted environments. While previous work has focused on mechanisms of short-term endocrine disruption and ovotoxicity in response to AHR ligands, we have shown recently that chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces premature reproductive senescence in female rats without depletion of ovarian follicular reserves. In the current study, premature reproductive senescence was induced using a range of low-dose exposure to TCDD (0, 1, 5, 50, and 200 ng kg(-1) wk(-1)) beginning in utero and continuing until the transition to reproductive senescence. Doses of 50 and 200 ng TCDD kg(-1) wk(-1) delayed the age at vaginal opening and accelerated the loss of normal reproductive cyclicity with age without depletion of follicular reserves. Serum estradiol concentrations were decreased in a dose-dependent fashion (>/=5 ng kg(-1) wk(-1)) across the estrous cycle in perisenescent rats still displaying normal cyclic vaginal cytology. Serum FSH, LH, and progesterone profiles were unchanged by TCDD. The loss of reproductive cyclicity following chronic exposure to TCDD was not accompanied by decreased responsiveness to GnRH. Ovarian endocrine disruption is the predominant functional change preceding the premature reproductive senescence induced by chronic exposure to low doses of the AHR-specific ligand TCDD.     

Effects of acute and chronic exposure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin on the transition to reproductive senescence in female Sprague-Dawley rats

Activation of the aryl hydrocarbon receptor (AHR) can occur in polluted environments, either from smoking-related toxicants or from endogenous ligands. We tested whether acute or chronic exposure to the AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the transition to reproductive senescence in female Sprague-Dawley rats. In experiment 1, rats (n = 6 per experimental group) received a single dose of 0 or 10 mug/kg of TCDD orally (p.o.) on Postnatal Day 29. Vaginal cytology was monitored for 1 wk each month until rats were killed at 1 yr of age. The single prepubertal exposure to TCDD hastened the transition to reproductive senescence in female rats and was associated with delayed puberty, abnormal cyclicity, and premature reproductive senescence. In a second experiment, rats were exposed to TCDD chronically through weekly dosing (0, 50, or 200 ng kg(-1) wk(-1) p.o., n = 7 each dose) beginning in utero. Lifelong exposure to these lower doses of TCDD induced a dose- and time-dependent loss of normal cyclicity and significantly hastened the onset of the transition to reproductive senescence (P < 0.05). This premature transition to reproductive senescence was associated with prolonged estrous cycles and, at the highest dose of TCDD, persistent estrus or diestrus. The number and size of ovarian follicles were not altered by TCDD. Diestrous concentrations of LH in rats exposed chronically to TCDD were similar to those in controls, whereas progesterone tended to be elevated at both doses of the dioxin (P < 0.08). Serum FSH was elevated in the group exposed to 50 ng/kg of TCDD (P < 0.02), whereas estradiol was decreased at both doses of dioxin (P < 0.01). Data thus far support endocrine disruption rather than depletion of follicular reserves as a primary mechanism of the premature transition to reproductive senescence following activation of the AHR pathway by TCDD in female rats.     

Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain

The aryl hydrocarbon receptor repressor (AHRR) is a negative regulator of AH receptor (AHR), which mediates most of the toxic and biochemical effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR has been shown to be the major reason for the exceptionally wide (ca. 1000-fold) sensitivity difference in acute toxicity of TCDD between two rat strains, sensitive Long-Evans (Turku/AB) (L-E) and resistant Han/Wistar (Kuopio) (H/W), but there is another, currently unknown contributing factor involved. In the present study, we examined AHRR structure and expression in these rat strains to find out whether AHRR could be this auxiliary factor. Molecular cloning of AHRR coding region showed that consistent with AHRR proteins in other species, the N-terminal end of rat AHRR is highly conserved, but PAS B and Q-rich domains are severely truncated or lacking. Identical structures were recorded in both strains. Next, the time-, dose-, and tissue-dependent expression of AHRR was determined using quantitative real-time RT-PCR. In liver, AHRR expression was very low in untreated rats, but it increased rapidly after TCDD exposure (100microg/kg). Testis exhibited the highest constitutive expression of AHRR, whereas kidney, spleen, and heart showed the highest induction of AHRR in response to TCDD treatment. Again, no marked differences were found between H/W and L-E rats, implying that AHRR is not the auxiliary contributing factor to the strain difference in TCDD sensitivity. However, simultaneous measurement of CYP1A1 mRNA reinforced the view that AHRR is an important determinant of tissue-specific responsiveness to TCDD.     

Treatment of mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin leads to aryl hydrocarbon receptor-dependent nuclear translocation of NF-kappaB and expression of Fas ligand in thymic stromal cells and consequent apoptosis in T cells

We investigated the role of aryl hydrocarbon receptor (AhR) in the regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis in thymic T cells. AhR knockout (KO) mice were resistant to TCDD-induced thymic atrophy and apoptosis when compared with the AhR wild-type mice. TCDD triggered the expression of several apoptotic genes, including FasL in AhR wild-type but not AhRKO mice. TCDD-induced increase in FasL was seen only in thymic stromal but not thymic T cells. When TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas-FasL interactions. Thus, apoptosis in T cells was not detected when TCDD-treated stromal cells from FasL-defective or AhRKO mice were mixed with wild-type T cells or when TCDD-exposed wild-type stromal cells were mixed with Fas-deficient T cells. TCDD treatment, in vivo and in vitro, led to colocalization and translocation of NF-kappaB subunits (p50, p65) to the nucleus in stromal but not T cells from AhR wild-type mice. NF-kappaB activation was not observed in stromal cells isolated from TCDD-treated AhRKO mice. Mutations in NF-kappaB-binding sites on the FasL promoter showed that TCDD regulates FasL promoter activity through NF-kappaB. TCDD treatment in vivo caused activation of the death receptor and mitochondrial pathways of apoptosis. Cross-talk between the two pathways was not necessary for apoptosis inasmuch as TCDD-treated Bid KO mice showed thymic atrophy and increased apoptosis, similar to the wild-type mice. These findings demonstrate that AhR regulates FasL and NF-kappaB in stromal cells, which in turn plays a critical role in initiating apoptosis in thymic T cells.     

Ah receptor in primate liver: binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and carcinogenic aromatic hydrocarbons

Ah receptor in hepatic cytosols from adult cynomolgus monkeys (Macaca fasicularis) was identified and quantitated by its binding of the highly toxic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the carcinogens 3-methylcholanthrene, benzo[a]pyrene, and dibenz[a,h]anthracene. The concentration of Ah receptor in cynomolgus hepatic cytosols (approximately 10 fmol/mg cytosol protein) was about one-quarter of that typically detected in rodent hepatic cytosols. Receptor concentrations were equal in male and female cynomolgus. [3H]TCDD bound to cytosolic receptor with high affinity (Kd approximately 3 nM). In rodents, Ah receptor is known to play a central role in toxicity caused by halogenated aromatic compounds and in carcinogenesis caused by polycyclic aromatic hydrocarbons. Existence of Ah receptor in monkeys indicates that the receptor also may mediate such responses in primates.     

Ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver, lung, and thymus

The ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in Sprague-Dawley rats by quantitation of the receptor in hepatic, lung, and thymic cytosol. Concentrations of hepatic and lung cytosolic receptors increased rapidly after birth and remained at the highest levels from days 2 to 21. After this time, receptor levels in these tissues slowly declined with age. In the thymus, cytosolic receptor concentrations remained high from days 2 to 42 following birth. In these tissues and at all times examined, the receptors demonstrated very high affinities for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. From days 15 to 42 following birth, no consistent sex related differences in receptor content or affinity were observed in any of these tissues.     

Physicochemical characterization of the nuclear form of Ah receptor from mouse hepatoma cells exposed in culture to 2,3,7,8-tetrachlorodibenzo-p-dioxin

Molecular properties of nuclear aromatic hydrocarbon (Ah) receptor from Hepa-1c1c9 (Hepa-1) cells were assessed by velocity sedimentation on sucrose gradients and by gel permeation chromatography on Sephacryl S-300. Nuclear Ah receptor was obtained by exposing intact cells to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 1 h at 37 degrees C in culture followed by extraction of receptor from nuclei with buffers containing 0.5 M KCl. The nuclear Ah receptor was compared to the cytosolic Ah receptor from the same cells. Under conditions of low ionic strength, the Ah receptor from Hepa-1 cytosol sedimented as a single 9.4 +/- 0.63 S binding peak that had a Stokes radius of 7.1 +/- 0.12 nm and an apparent relative molecular mass of 271,000 +/- 16,000. After prolonged (24 h) exposure to high ionic strength (0.5 M KCl), cytosol labeled with [3H]TCDD exhibited two specific binding peaks. The large form of cytosolic Ah receptor seen under high ionic strength conditions sedimented at 9.4 +/- 0.46 S, had a Stokes radius of 6.9 +/- 0.19 nm, and an apparent Mr 267,000 +/- 15,000. The smaller ligand-binding subunit generated by exposing cytosol to 0.5 M KCl sedimented at 4.9 +/- 0.62 S, had a Stokes radius of 5.0 +/- 0.14 nm, and an apparent Mr 104,000 +/- 12,000. Nuclear Ah receptor, analyzed under high ionic strength conditions, sedimented at 6.2 +/- 0.20 S, had a Stokes radius of 6.8 +/- 0.19 nm, and an apparent Mr 176,000 +/- 7000. Nuclear Ah receptor from rat H4IIE hepatoma cells was analyzed and found to have physicochemical characteristics identical to those of nuclear Ah receptor from the mouse Hepa-1 cells. The molecular mass of Hepa-1 nuclear Ah receptor was found to be statistically different from both the Mr approximately 267,000 cytosolic Ah receptor and the Mr approximately 104,000 subunit which were present in cytosol under high ionic strength conditions. Hepa-1 nuclear Ah receptor could not be converted to a smaller ligand-binding subunit by treatment with alkaline phosphatase, ribonuclease, or sulfhydryl-modifying reagents or prolonged exposure to 1.0 M KCl. Cytosolic Ah receptor from Hepa-1 cells was "transformed" by heating at 25 degrees C in vitro into a form with high affinity for DNA-cellulose. The transformed cytosolic Ah receptor, when analyzed under conditions of high ionic strength, sedimented at approximately 6 S, had a Stokes radius of approximately 6.7 nm, and an apparent Mr approximately 167,000.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of thyroidectomy on the Ah receptor and enzyme inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat liver

Thyroidectomy of rats confers some protection, by an unknown mechanism, from the weight loss, immunotoxicity, and mortality induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since at least some of the many effects of TCDD appear to be mediated by the Ah receptor, perhaps the thyroid plays a role in regulation of this receptor, thereby modifying the toxicity of TCDD. We tested this hypothesis by comparing TCDD-binding characteristics of the receptor and hepatic enzyme inducibility by TCDD (a receptor-mediated response) in thyroidectomized (ThX) and euthyroid rats. There were no significant differences in levels of TCDD binding in vitro in hepatic cytosol, in receptor affinity, nor in the molecular size of the TCDD-bound receptor in untreated ThX rats compared to controls fed ad libitum or pair-fed. Total hepatic cytochrome P-450 (P-450) levels and NADPH-menadione oxidoreductase (NMOR) activity were unaffected by thyroid status, whereas 7-ethoxycoumarin O-deethylase (ECOD) activity was approx. 50% lower in ThX animals than in ad libitum or pair-fed controls. At 3 and 10 days after TCDD administration (10 micrograms/kg, i.p.), P-450 concentrations and NMOR and ECOD activities were induced by approximately the same proportions in ThX and pair-fed intact rats; however, the absolute levels of the induced activities were lower in ThX than in pair-fed controls. It was concluded that hypothyroidism does not regulate Ah receptor concentration or function in the liver. Therefore, the modulation of TCDD toxicity by hypothyroidism appears not to involve changes in the hepatic Ah receptor.     

芳香烃受体核转位蛋白的结构及相关功能

芳香烃受体核转位蛋白（aryl hydrocarbon receptor nuclear translocator,ARNT）是碱性螺旋-环．螺旋转录因子超家族中新发现的PAS亚家族的成员之一。它是体内许多bHLH-PAS蛋白共同的专性配偶体,可以与芳香烃受体、低氧诱导因子、果蝇SIM蛋白等形成异二聚体并介导许多信号转导过程,从而使个体对环境污染物（如二恶英）、低氧状态等外界因素的改变产生相应的生物学效应,本文就ARNT的基本结构及其在体内的主要生理功能等方面作一综述。     

Cutting edge: activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin generates a population of CD4+ CD25+ cells with characteristics of regulatory T cells

Activation of the aryl hydrocarbon receptor (AhR) by its most potent ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to immune suppression in mice. Although the underlying mechanisms responsible for AhR-mediated immune suppression are not known, previous studies have shown that activation of the AhR must occur within the first 3 days of an immune response and that CD4+ T cells are primary targets. Using the B6-into-B6D2F1 model of an acute graft-vs-host response, we show that activation of AhR in donor T cells leads to the generation of a subpopulation of CD4+ T cells that expresses high levels of CD25, along with CD62L(low), CTLA-4, and glucocorticoid-induced TNFR. These donor-derived CD4+ CD25+ cells also display functional characteristics of regulatory T cells in vitro. These findings suggest a novel role for AhR in the induction of regulatory T cells and provide a new perspective on the mechanisms that underlie the profound immune suppression induced by exposure to TCDD.