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.     

Involvement of the xenobiotic response element (XRE) in Ah receptor-mediated induction of human UDP-glucuronosyltransferase 1A1

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by contributing to the metabolism of endogenous substances such as bilirubin in addition to xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear receptors steroid xenobiotic receptor (SXR) and the constitutive active receptor, CAR. In this report, we show that in human hepatoma HepG2 cells the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah receptor) ligands such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), beta-naphthoflavone, and benzo[a]pyrene metabolites. Induction was monitored by increases in protein and catalytic activity as well as UGT1A1 mRNA. To examine the molecular interactions that control UGT1A1 expression, the gene was characterized and induction by Ah receptor ligands was regionalized to bases -3338 to -3287. Nucleotide sequence analysis of this UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at -3381/-3299. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by a loss of Ah receptor ligand inducibility when the XRE core region (CACGCA) was deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah receptor and Arnt antibodies demonstrated that the nuclear protein was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.     

Use of c-Src and c-Fos knockout mice for the studies on the role of c-Src kinase signaling in the expression of toxicity of TCDD

Previously, we reported that several of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p -dioxin (TCDD) were not fully expressed in c-src −/− and −/+ mice (1). In the current study, we studied the basic molecular mechanism of their differential responses. First, we could show that chemical inhibition of c-src kinase could produce practically the same phenomenon of reduced toxicity of TCDD in wild-type mice cotreated with geldanamycin, a specific chemical inhibitor known to suppress c-src kinase. Second, we established that the level of the Ah receptor associated with c-src kinase (2) is indeed low in c-src deficient mice as well as geldanamycin-treated mice. We could show, at the same time, that the effect of c-src deficiency on the toxicity of TCDD is very selective; that is, despite the reduction of many of its toxic signs, the enlargement of liver, induction of cytochrome P450, and other drug-metabolizing enzymes took place normally in those c-src–deficient mice. Apparently, induction of these detoxification enzymes are independent of c-src–mediated pathway. Based on the known signaling pathways of c-src, we tested c-fos–deficient mice and found that some of the c-src–dependant toxic signs of TCDD such as thymic atrophy and decrease in adipose tissue weight were also reduced in c-fos–deficient mice, indicating that these two toxic effects are likely to be mediated through both c-src and c-fos. However, other TCDD responses noticeable in c-fos–deficient mice; downregulation of receptors for epidermal growth factor (EGF), tumor necrosis factor (TNFα), and retinoic acid; and up-regulation of the T3 receptor. These findings clearly show that c-fos mediates only a part of c-src signaling pathway in transducing these specific toxic actions of TCDD as mediated by c-fos. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 263–274, 1998     

Characterization of the Ah receptor mediating aryl hydrocarbon hydroxylase induction in the human liver cell line Hep G2

The Ah receptor, a soluble cytoplasmic receptor that regulates induction of cytochrome P450IA1 and mediates toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was detected and characterized in the continuous human liver cell line Hep G2. The mean concentration of specific binding sites for TCDD was 112 +/- 26 (SEM) fmol/mg cytosol protein as determined in eight separate cytosol preparations in the presence of sodium molybdate. This is equivalent to 14,000 binding sites per cell, approximately 40% of the sites per cell found in the mouse hepatoma line Hepa-1. The cytosolic Ah receptor from Hep G2 cells sedimented at 9 S and was specific for those halogenated and nonhalogenated aromatic compounds known to be agonists for the Ah receptor in rodent tissues and cells. Specific binding in the 9 S region was detected with both [3H]TCDD and 3-[3H]methylcholanthrene. 3-[3H]Methylcholanthrene did not bind to any component besides that at approximately 9 S. Phenobarbital, dexamethasone, and estradiol did not compete with [3H]TCDD for binding to the Hep G2 Ah receptor. Specific binding of [3H]triamcinolone acetonide to glucocorticoid receptor could also be demonstrated in Hep G2 cytosol. The apparent equilibrium dissociation constant (Kd) for binding of [3H]TCDD to Hep G2 Ah receptor was 9 nM by Woolf plot analysis, about an order of magnitude weaker than the affinity of [3H]TCDD for the mouse Hepa-1 Ah receptor or for the C57BL/6 murine hepatic Ah receptor. [3H]TCDD.Ah receptor complex, which was extracted from nuclei of Hep G2 cells incubated with [3H]TCDD at 37 degrees C in culture, sedimented at approximately 6 S under conditions of high ionic strength. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced after 24 h of incubation with polycyclic aromatic hydrocarbons: the EC50 for AHH induction was 5.3 microM for benz(a)anthracene and 1.3 microM for 3-methylcholanthrene. Modification of the preparative technique for cell cytosol, especially inclusion of 20 mM sodium molybdate in homogenizing and other buffers, was necessary to detect cytosolic Hep G2 Ah receptor. Hep G2 cells appear to conserve drug-metabolizing activity associated with cytochrome P450IA1 as well as the receptor mechanism which regulates its induction.     

Ah receptor in spleen of rodent and primate species: detection by binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin

In many species systemic toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is manifested by a generalized wasting syndrome accompanied by a variety of specific organ changes including atrophy of the thymus and spleen. TCDD toxicity in most tissues is thought to be mediated by the Ah receptor. Although the spleen is a prime target for TCDD toxicity, the possible presence of Ah receptor in the spleen has not previously been investigated. Specific binding of [3H]TCDD to Ah receptor in spleen cytosols was assessed by velocity sedimentation on sucrose gradients. Ah receptor was detected in spleen cytosols from adult Rhesus monkeys (mean +/- SEM, 36 +/- 8 fmol/mg cytosol protein), fetal Rhesus monkeys (9 +/- 6), Sprague-Dawley rats (20 +/- 5), C57BL/6J mice (18 +/- 2), New Zealand white rabbits (19 +/- 2), and Hartley guinea pigs (15 +/- 2). Ah receptor was not detectable in spleen cytosol from genetically "nonresponsive" DBA/2J mice or from Golden Syrian hamsters, a species resistant to toxicity of TCDD. Molecular properties of Ah receptor from spleen were similar to those of the receptor from liver of the same species. The high Ah receptor content in spleen cytosols from those species that are most susceptible to TCDD toxicity is consistent with the view that the Ah receptor mediates TCDD toxicity in spleen as well as in other tissues.     

Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Evidence that hexachlorobenzene is a weak Ah receptor agonist

Hexachlorobenzene (HCB) produces hepatic porphyria and induces the hepatic cytochrome P450 isozymes P450c (P450IA1) and P450d (P450IA2) in rodents. These and other effects of HCB resemble those of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which acts via its binding to the aromatic hydrocarbon (Ah) receptor. We therefore examined the ability of HCB to interact with this receptor in vitro and in vivo. HCB, at concentrations of 1 microM or higher, inhibited the specific binding of [3H]TCDD (0.3 nM) to the Ah receptor in vitro, whereas the solubility of [3H]TCDD was affected only at 100 microM HCB. The inhibition was competitive, with a KI of approximately 2.1 microM. In rats fed a diet containing 3000 ppm HCB for varying times (4 h to 7 days), the specific binding of [3H]TCDD in hepatic cytosol was reduced by up to 40%, as observed previously for known Ah receptor agonists. The decrease in [3H]TCDD specific binding in cytosol of HCB-treated rats was due principally to a decrease in the number of binding sites for [3H]TCDD rather than competition from residual HCB. As shown by immunoblotting and radioimmunoassay, HCB induced the cytochrome P450 isozymes P450c and P450d, which are regulated by the Ah receptor, as well as the phenobarbital-inducible isozymes P450b and P450e. Together these results indicate that HCB is a weak agonist for the Ah receptor, and suggest that some of its effects may be mediated by its interaction with this gene-regulatory protein.     

Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: ontogeny in chick embryo liver

Aryl hydrocarbon hydroxylase (AHH, cytochrome P1-450) is induced in chick liver very early during embryonic development if embryos are treated with 3-methylcholanthrene-type compounds such as 3,4,3'4'-tetrachlorobiphenyl. In mammals, AHH induction is known to be mediated by the Ah receptor. Liver from embryonic and newly hatched chicks was found to contain a cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has properties that are very similar to properties of the Ah receptor previously characterized in mammalian tissues. In chick embryo liver, cytosolic binding sites for TCDD were of high affinity (Kd for [3-H]-TCDD = 0.2 nM) and were specific for 3-methylcholanthrene-type inducers. The specific binding component sedimented at about 9S on sucrose density gradients prepared at low ionic strength. A high level of Ah receptor was detected in chick embryo liver by the fifth day of incubation (5 DI); this is at least 24 hours prior to the onset of AHH inducibility. The Ah receptor concentration increased from 5 DI to 8 DI, the period when chick liver is undergoing early morphological differentiation. After 8 DI, Ah receptor levels dropped substantially and remained low into the posthatching period. In contrast, AHH inducibility was high by 7 DI and remained high throughout embryonic development and into the posthatching period. The discrepancy between Ah receptor levels and the degree of AHH inducibility suggests that only a small fraction of the Ah receptor population is required for maximal AHH induction.     

Poxvirus protein N1L targets the I-kappaB kinase complex, inhibits signaling to NF-kappaB by the tumor necrosis factor superfamily of receptors, and inhibits NF-kappaB and IRF3 signaling by toll-like receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.