Analysis of Ah receptor-ARNT and Ah receptor-ARNT2 complexes in vitro and in cell culture.

ARNT and ARNT2 proteins are expressed in mammalian and aquatic species and exhibit a high level of amino acid identity in the basic-helix loop-helix PER/ARNT/SIM domains involved in protein interactions and DNA binding. Since the analysis of ARNT2 function at the protein level has been limited, ARNT2 function in aryl hydrocarbon receptor (AHR)-mediated signaling was evaluated and compared to ARNT. In vitro, ARNT and ARNT2 dimerized equally with the AHR in the presence of 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) and ARNT2 outcompeted ARNT for binding to the AHR when expressed in excess. In contrast, activation of the AHR with 3-methylcholanthrene or benzo[a]pyrene resulted in predominant formation of AHR*ARNT complexes. ARNT2 expressed in Hepa-1 cell culture lines with reduced ARNT protein resulted in minimal induction of endogenous CYP1A1 protein compared to cells expressing ARNT, and mutation of the putative proline residue at amino acid 352 to histidine failed to produce an ARNT2 that could function in AHR-mediated signaling. However, the expression of ARNT2 in wild-type Hepa-1 cells reduced TCDD-mediated induction of endogenous CYP1A1 protein by 30%, even though AHR*ARNT2 complexes could not be detected in nuclear extracts. Western blot analysis of numerous mouse tissues and various cell culture lines showed that both endogenous ARNT and ARNT2 could be detected in cells derived from kidney, central nervous system, and retinal epithelium. Thus, ARNT2 has the ability to dimerize with the liganded AHR in vitro and is influenced by the activating ligand yet appears to be limited in its ability to influence AHR-mediated signaling in cell culture.     

Correlation of cardiotoxicity mediated by halogenated aromatic hydrocarbons to aryl hydrocarbon receptor activation.

In mammals, the toxicity of halogenated aromatic hydrocarbons (HAH) correlates with their ability to activate the aryl hydrocarbon receptor (AHR). To test this correlation in an avian model, we selected six HAHs based on their affinity for the mammalian AHR, including: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PCDD); 2,3,7,8-tetrachlorodibenzofuran (TCDF); 2,3,4,7,8-pentachlorodibenzofuran (PCDF); 3,3',4,4'-tetrachlorobiphenyl (PCB 77); and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). We determined the ability of these compounds to induce cardiotoxicity, as measured by an increase in heart wet weight on incubation day 10 in the chick embryo (Gallus gallus) and formation of the avian AHR/ARNT/DNA binding complex in chicken hepatoma cells. Relative potency values (RPs) were calculated by dividing the TCDD EC(50) (AHR/ARNT/DNA binding) or ED(50) (15% increase in day-10 heart wet weight) by the HAH congeners EC(50) or ED(50), respectively. The rank order of potencies for inducing cardiotoxicity were TCDD > PCDD = PCDF = TCDF > PCDF > PCB77, PCB 153, no effect. The RP values for inducing AHR/ARNT DNA binding were then correlated with those for inducing cardiotoxicity (the RP values of PCDD were determined to be statistical outliers). This correlation was found to be highly significant (r = 0.94, p = 0.017). The ability of PCDD to act as an AHR agonist was verified using luciferase reporter assays and analysis of cytochrome P4501A1 protein levels. These results indicate that the ability of HAHs to activate the avian AHR signaling pathway, in general, correlates with their ability to mediate cardiotoxicity in the chick embryo.     

Repression of aryl hydrocarbon receptor (AHR) signaling by AHR repressor: role of DNA binding and competition for AHR nuclear translocator

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving "transrepression" of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.     

Female Sprague--Dawley Rats Exposed to a Single Oral Dose of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exhibit Sustained Depletion of Aryl Hydrocarbon Receptor Protein in Liver, Spleen, Thymus, and Lung

There is currently little information concerning the time-dependentrelationship between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)exposure and aryl hydrocarbon receptor (AHR) and aryl hydrocarbonreceptor nuclear translocator (ARNT) protein concentration invivo. Therefore, female Sprague-Dawley rats were given a singleoral dose of TCDD (10 µg/kg), and the AHR and ARNT proteinconcentrations in liver, spleen, thymus, and lung determinedby Western blotting. In liver, the concentration of AHR proteinwas significantly reduced 8 and 24 h postdosing as comparedto time-matched controls. In spleen and lung, the concentrationof AHR protein was reduced 3, 8, 24, and 168 h posttreatmentcompared to time-matched controls but returned to control levelsby 336 h. In thymus, reductions in AHR protein concentrationwere observed 8, 24, 168, and 336 h postdosing as compared totime-matched controls. Significant reductions in the concentrationof ARNT protein were not observed in any of the TCDD-exposedtissues. Functional studies in cell culture showed that exposureof a mouse hepatoma cell line (Hepa-1c1c7) and a rat smoothmuscle cell line (A-7) to TCDD (1 nM) for 12 days resulted ina 50% reduction in TCDD-inducible reporter gene expression followingsubsequent challenge by an additional dose of TCDD (1 nM). Collectively,these results show that (i) TCDD-mediated depletion of AHR occursin vivo, (ii) AHR protein does not rapidly recover to pretreatmentlevels even though the tissue concentration of TCDD has fallen,and (iii) reduction in AHR protein concentration correlateswith reduction in TCDD-mediated reporter gene expression inmammalian culture cells.     

Analysis of rainbow trout Ah receptor protein isoforms in cell culture reveals conservation of function in Ah receptor-mediated signal transduction

Two distinct aryl hydrocarbon receptor (AHR) cDNAs have been isolated from rainbow trout. The encoded receptor protein products termed rtAHR2alpha and rtAHR2ss are 97% identical at the amino acid level but are reported to have distinct functions with regard to AHR-mediated gene regulation. To test this hypothesis, the two proteins were evaluated functionally both in vitro and in a Chinese hamster lung cell line, E36. To facilitate analysis, both rtAHR2 isoforms were tagged with the FLAG peptide and could be expressed and quantified in a rabbit reticulocyte lysate. However, both proteins failed to form functional complexes with mammalian or rainbow trout AHR nuclear translocator protein (ARNT) that could associate with xenobiotic response elements (XREs) in a ligand-dependent manner in vitro. In contrast, both proteins exhibited positive function on AHR-mediated signaling when expressed in the E36 cell line. Both rtAHR2 isoforms showed a cytoplasmic distribution in the unliganded state and could drive the expression of a reporter gene under control of the trout CYP1A3 promoter. Although both proteins induced reporter gene activity to the same magnitude, the EC(50) values of the two isoforms for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) differed by an order of magnitude, with the rtAHR2ss isoform less responsive to TCDD. When the functions of the rtAHR2 isoforms were tested in the context of the dominant negative rtARNT(a) protein, TCDD-mediated induction of reporter gene activity was reduced as the level of rtARNT(a) protein increased. In summary, both rtAHR2 isoforms appear to exhibit positive function in AHR-mediated signaling, suggesting conservation of function.     

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the expression of cytochrome P450 1A1, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator in rat brain and pituitary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants causing a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and neuroendocrine-related organs including the pituitary gland. However, so far there is little evidence for direct effects of TCDD on these areas. In the present study, male Sprague-Dawley rats were treated with a single oral dose of TCDD (10 microg/kg) and euthanized 1, 3, or 28 days after treatment. The expression of cytochrome P450 1A1 (CYP1A1), the aryl hydrocarbon receptor (AHR), and the aryl hydrocarbon receptor nuclear translocator (ARNT) were analyzed in different brain regions and pituitaries using semiquantitative RT-PCR and Western blotting. Relative levels of CYP1A1 mRNA and protein were dramatically increased in the pituitary. A significant increase in CYP1A1 mRNA was also detected in all the brain regions examined including olfactory bulb, striatum-caudate, hypothalamus, hippocampus, cortex, cerebellum, and substantia nigra. The increase in the expression was time-dependent with the highest level observed 1 day after TCDD treatment. The AHR and ARNT mRNAs were detected in the same areas but in contrast to CYP1A1 the changes in AHR and ARNT mRNA expression were limited to the 28-day time point. The present results provide evidence for the presence of CYP1A1, AHR, and ARNT in the central nervous system and in the pituitary, suggesting that TCDD may exert a direct effect on these regions.     

Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells

Changes in the expression of the aryl hydrocarbon receptor (AHR) have been documented in several systems and in response to a variety of treatments. The significance of these findings is unclear, because the effects of such changes on subsequent responses to AHR ligands seldom have been measured. We tested the ability of changes in serum used in cell culture medium to alter expression of the AHR and induction of cytochrome P4501A (CYP1A) in PLHC-1 teleost hepatoma cells. Culture of early-passage cells in serum-free medium for 2 days led to a loss of CYP1A inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, culture in 10% delipidated calf serum increased the TCDD-induced levels of both CYP1A protein and enzymatic activity relative to levels in cells cultured in 10% complete calf serum. These effects were consistent between 8 and 24hr post-treatment, indicating that the kinetics of induction were unaffected. In cells cultured in serum-free medium for 1 and 2 days there was a progressive loss of CYP1A inducibility. This loss of response paralleled a time-dependent decline in AHR protein, as measured by specific binding of [3H]TCDD. Using an operational model for AHR action in PLHC-1 cells, the measured reduction in AHR could be shown to predict the loss of CYP1A induction. Expression of AHR protein was unaffected by culture in 10% delipidated serum. The effects of serum-free medium and delipidated serum were found only in early-passage cells; inducibility of CYP1A and expression of AHR protein in late-passage cells were unaffected by serum withdrawal. Comparison of early- and late-passage cells revealed a 2-fold greater rate of proliferation in the latter, suggesting that a growth advantage is coincident with loss of the serum-dependency of AHR expression. These results provide a quantitative link between changes in receptor expression and a downstream response, establishing a foundation for future studies of receptor expression and sensitivity to toxic responses in vitro and in vivo.     

Aryl hydrocarbon receptor activation inhibits regenerative growth

There is considerable literature supporting the conclusion that inappropriate activation of the aryl hydrocarbon receptor (AHR) alters cellular signaling. We have established previously that fin regeneration is specifically inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult zebrafish and have used this in vivo endpoint to evaluate interactions between AHR and growth-controlling pathways. Because there are experimental limitations in studying regeneration in adult animals, we have developed a larval model to evaluate the effect of AHR activation on tissue regeneration. Two-day-old zebrafish regenerate their amputated caudal fins within 3 days. Here, we demonstrate that TCDD specifically blocks regenerative growth in larvae. The AHR pathway in zebrafish is considerably more complex than in mammals, with at least three zebrafish AHR genes (zfAHR1a, zfAHR1b, and zfAHR2) and two ARNT genes (zfARNT1 and zfARNT2). Although it was presumed that the block in regeneration was mediated by AHR activation, it had not been experimentally demonstrated. Using antisense morpholinos and mutant fish lines, we report that zfAHR2 and zfARNT1 are the in vivo dimerization partners that are required for inhibition of regeneration by TCDD. Several pathways including fibroblast growth factor (FGF) signaling are essential for fin regeneration. Even though impaired FGF signaling and TCDD exposure both inhibit fin regeneration, their morphometric response is distinct, suggesting that the mechanisms of impairment are different. With the plethora of molecular and genetic techniques that can be applied to larval-stage embryos, this in vivo regeneration system can be further exploited to understand cross-talk between AHR and other signaling pathways.     

An aryl hydrocarbon receptor odyssey to the shores of toxicology: the Deichmann Lecture, International Congress of Toxicology-XI.

The science of toxicology is devoted, in large part, to understanding mechanisms of toxicity so that we can more accurately assess the risk posed by exposure to xenobiotic agents and, perhaps, intervene in the toxicologic process to mitigate harm. Dioxin-like chemicals continue to be of great concern as environmental toxicants. About 30 years ago the aryl hydrocarbon receptor (AHR) was discovered as a specific binding site for 2,3,7,8-tetrachlorodibenzo-p-dioxin. This giant step led to our current view that essentially all toxic effects of dioxins are AHR-mediated. The AHR serves as the archetype for understanding toxicity mediated by other soluble receptors. The fact that toxicity is receptor-mediated has important implications, especially for dose-response relationships. In laboratory animals genetic differences in AHR gene structure lead to profound differences in responsiveness to dioxin-like chemicals. Humans, however, exhibit relatively few AHR polymorphisms and these seem to exert only modest effects on downstream events. Dioxin toxicity is fundamentally due to AHR-mediated dysregulation of gene expression. Our current challenging goal is to determine which dysregulated genes underlie specific forms of dioxin toxicity. Mapping AHR-mediated gene expression in a variety of biological systems may help explain why dramatic differences in susceptibility to dioxin toxicity exist among laboratory species and why humans appear to be relatively resistant to adverse effects of dioxins.     

Persistent, low-dose 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: effect on aryl hydrocarbon receptor expression in a dioxin-resistance model

Most toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the aryl hydrocarbon receptor (AHR). A single, acute dose of TCDD can alter its own receptor levels thus complicating evaluation of dose-response relationships for AHR-mediated events. Since environmental exposure to dioxins is typically of a repeated low-dose nature, we examined the effect of such exposure on AHR expression. Three rat strains differing greatly in their sensitivity to acute TCDD lethality, Long-Evans (Turku AB) (L-E) (LD50 approximately 10 microg/kg); Sprague Dawley (SD) (LD50 approximately 50 microg/kg); and Han/Wistar (Kuopio) (H/W) (LD50 > 9600 microg/kg), were administered TCDD intragastrically, biweekly for 22 weeks producing doses equivalent to 0, 10, 30, and 100 ng/kg/day. Changes in hepatic AHR levels were quantitated at the protein level by radioligand binding and immunoblotting and at the mRNA level by RT-PCR. Cytosolic AHR protein was elevated at 10 or 30 ng/kg/day TCDD in SD and L-E rats; AHR mRNA was also elevated at these doses, suggesting a pretranslational mechanism. There was no apparent relationship between TCDD-induced AHR regulation and strain sensitivity to TCDD. Overall, "subchronic" TCDD did not greatly perturb AHR expression. The maintenance of relatively constant receptor levels in the face of persistent agonist stimulation is in contrast to the sustained depletion of AHR by TCDD observed in cell culture and to the fluctuations in AHR observed hours to days following acute TCDD exposure in vivo. Changes in AHR levels may affect dose-response relationships; the effect of TCDD on its own receptor at environmentally relevant dosing schemes is therefore important to risk assessment.