AH RECEPTOR STRUCTURE AND DIOXIN SENSITIVITY

Project leader: Raimo Pohjanvirta, University of Helsinki, Faculty of Veterinary Medicine Department of Food and Environmental Hygiene, Helsinki, Finland & National Veterinary and Food Research Institute, Kuopio Department, Kuopio, Finland.
Contact address: EELA, P.O.Box 92, FIN-70701 Kuopio, Finland, tel. +358-50-5469019, e-mail: Raimo.Pohjanvirta@eela.fi
 
 

PUBLICATIONS

TIIVISTELMÄ SUOMEKSI

Researcher:
Merja Korkalainen, National Public Health Institute, Laboratory of Toxicology, Kuopio, Finland, tel. +358-17-201318, e-mail: Merja.Korkalainen@ktl.fi

Consortium: Environmental health risks of dioxins
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 111306 / 220628
Person-months of work funded by SYTTY / Total person-months of work: 28 / 147

KEY WORDS: AH receptor, dioxin, TCDD
 

EXTENDED ABSTRACT

1 Introduction

Dioxins and related halogenated aromatic hydrocarbons are ubiquitous environmental contaminants. Evaluation of the risks posed by these compounds to humans is hampered by the exceptionally large inter- and intraspecies differences occurring in laboratory animals for some of their effects. These differences culminate in acute lethality: for the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the hamster is about 1000-fold more resistant than the guinea pig and a difference of the same magnitude exists between a TCDD-sensitive rat strain, Long-Evans (Turku AB) [L-E] and a TCDD-resistant strain, Han/Wistar (Kuopio) [H/W]. By contrast, both H/W rats and hamsters are susceptible to, e.g., enzyme induction, thymus atrophy and fetotoxicity by TCDD.

Most of the biological effects of dioxins are mediated by a cytosolic protein called the AH (aryl hydrocarbon) receptor (AHR). The AHR is a ligand-activated transcription factor which structurally belongs to a newly discovered family of proteins: basic helix-loop-helix (bHLH)/PAS transcription factors. The molecular mechanism of its action has so far been only resolved for CYP1A1 induction, but this is believed to be a general mode of gene regulation by the AHR. In an inactive state, the AHR is located in the cytoplasm in association with two molecules of the chaperone hsp90, an immunophilin-like protein and possibly a few other proteins. Binding of ligand such as TCDD results in transformation of the receptor with translocation into the nucleus and shedding of the associated proteins. Inside the nucleus, AHR dimerizes with a related bHLH/PAS protein, ARNT, and then binds to DNA at specific sites containing a consensus hexanucleotide core. These dioxin response elements act as enhancers for genes regulated by dioxins. Since the enhancer sites are usually situated far upstream of the gene promoter, gene activation by dioxins probably involves nucleosomal disruption and interaction with transcriptional coactivators and/or corepressors.

The AHR protein consists of distinct functional modules. The bHLH domain located in the N terminus is responsible for DNA binding and heterodimerization. The PAS motif flanking the bHLH structure affords specificity to dimerization and also contains most of the ligand binding domain. The C terminus comprises a potent transactivation domain composed of several interacting subdomains, one of which is a glutamine-enriched (Q-rich) subunit.

The goal of this project was to gain further insight into the role of the AHR as a whole, and its transactivation domain in particular, in TCDD toxicity. Recent cloning of the H/W rat AHR cDNA by us disclosed three splice variants giving rise to two different proteins, both of which are smaller than the wild-type rat receptor and bear unique transactivation domains. Genetic studies implied that the altered AHR is the major determinant of TCDD resistance in H/W rats. These findings led us to postulate
that the peculiar patterns of selective responsiveness to TCDD in H/W rats and hamsters are largely due to the structure of their AHR transactivation domains. An additional objective was to measure to what extent TCDD regulates expression of its own receptor and whether this mechanism could contribute to rat strain differences in TCDD sensitivity.

2 Methods

Several independent but complementary approaches were pursued:

1) AHR cloning from hamster and guinea pig
Hamster and guinea pig AHR cDNAs were cloned using RT-PCR and blunt-end cloning with liver total RNA as starting material. The ends of the coding sequence were obtained by modified RACE techniques. The clones were sequenced with an automatic sequencer.

2) AHR transgenic mice
In mice, a venture was commenced to produce transgenic animals expressing only rat AHR variants. To this end, C57BL/6x129 hybrid mice were used in the pronuclear microinjection procedure in which one of the 3 rat AHR variants at a time was transferred into mouse zygotes. Pups harboring the transgene in their genomes were found by PCR analysis of the genomic DNA. The positive mice will next be crossed and backcrossed with AHR knockouts (purchased form Jackson Laboratory, USA)  in order to get rid of the endogenous murine AHR, and the level of rat-derived AHR expression will then be measured. If assessed sufficient, the mice will be tested and compared with one another for their TCDD responsiveness with a battery of toxicity endpoints. This study is underway at present.

3) Effect of TCDD on AHR expression
Three rat strains with widely different sensitivities to the acute lethality of TCDD were exposed either to a single dose (0, 5 or 50 µg/kg) or repeated doses (0, 10, 30 or 100 ng/kg/day for 22 weeks) of TCDD. Changes in hepatic AHR concentrations were quantitated at the protein level by radioligand binding and immunoblotting and at the mRNA level by semiquantitative RT-PCR. This study was performed in collaboration with Dr. A. B. Okey, Toronto, Canada.

4) Comparison of AHR variant function in vitro
The variant H/W rat AHR cDNAs will be expressed in a yeast cell culture to gain insight into their abilities to drive expression of a dioxin response element –containing reporter gene as well as into their interactions with transcriptional coactivators. Furthermore, composite artificial receptors with a heterologous DNA-binding protein Gal4 will be constructed of both the wild-type and variant rat AHR transactivation domains to enable direct comparison of their transactivation capabilities. These studies are underway at present in Stockholm, Sweden by Dr. L. Poellinger.

Finally, loosely connected with these studies, we examined effects of a “natural” AHR agonist (indolo[3,2-b]carbazole; ICZ) and an antagonist (resveratrol) at the whole animal level in L-E rats. Both compounds were given alone or with TCDD and a number of typical AHR-mediated responses were measured.

3 Results and discussion

Structure of hamster and guinea pig AHR
Analysis of the deduced primary structure of AHR revealed that the N-terminal end of the receptor was highly conserved in both species. However, there proved to be a conspicuous change in the C-terminus of the hamster AHR bearing the transactivation domain. In hamster, the Q-rich subdomain of the receptor was strikingly expanded with the number of glutamine residues being almost twice as high as in its mouse, rat and human counterparts. Expression of the AHR, as determined by semiquantitative RT-PCR, was similar in hamsters to that reported in rats with highest levels occurring in liver, lung and thymus. In guinea pig AHR, the number of glutamine residues in the Q-rich subdomain was notably small. Surprisingly, the closest homologue of the guinea pig AHR turned out to be the human AHR, which also has a low glutamine content in the Q-rich subdomain. It has previously been shown that this subdomain is functionally essential to the AHR, and that a change of even a single individual amino acid at a critical location in the Q-rich region can markedly modulate AHR function. Thus, the substantial alteration found in hamster AHR may well have a causal bearing on the exceptional resistance of this species to TCDD. Moreover, there appeared to be an inverse correlation across species between TCDD sensitivity and number of glutamine residues in the Q-rich subdomain, which is intriguing and might be relevant to human risk assessment.

Modulation of AHR expression by TCDD

In the acute experiment, the rats were euthanized at 1, 4 or 10 days after TCDD exposure. The lower dose tested (5 µg/kg; sublethal to all rats) produced a 2-3-fold increase in cytosolic AHR protein in all 3 strains; the higher dose (50 µg/kg; lethal to L-E and SD strains) elicited AHR protein decline on day 1 followed by recovery in SD and H/W but not in L-E rats. Both the increase in AHR protein above basal levels and the recovery from initial decrease were accompanied by elevations in steady-state AHR mRNA. The time frame for AHR upregulation was different from that of CYP1A1 induction suggesting different modes of regulation for these two genes. There was no clear relationship between AHR regulation and strain sensitivity; thus, the large strain differences in susceptibility to TCDD lethality are probably not explained by differential regulation of AHR by TCDD.

In the repeated dosing experiment, cytosolic AHR protein was elevated at the lowest doses (10 and 30 ng/kg/day TCDD) in SD and L-E rats; AHR mRNA was also increased at these doses suggesting a pretranslational mechanism. Overall, ”subchronic” TCDD exposure 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 recorded hours to days following acute TCDD exposure in vivo.

Activity of resveratrol and ICZ at the whole animal level

Resveratrol occurs in red wine and has aroused general interest due to its AHR-antagonistic properties under in vitro conditions. ICZ is formed in the gastrointestinal tract as a condensation product from indole-3-carbinol, which occurs in cruciferous vegetables such as cabbage. In vitro, it exhibits AHR agonist activity comparable to that of TCDD. Daily intake of ICZ is much higher than that of dioxins and could thus be a reason for concern. However, our studies in TCDD-sensitive L-E rats demonstrated that the ability of ICZ to bring about typical dioxin-like effects in vivo was remarkably low, plausibly due to a rapid biotransformation of the compound. Likewise, the very high dose of resveratrol tested did not interfere with TCDD action, nor did it have any discernible effects of its own. These studies underscored the importance of verifying preliminary in vitro findings at the whole animal level before drawing far-reaching conclusions regarding potential human risk.

4 Conclusions

The C-terminal transactivation domain seems to be an important determinant in the AHR structure of species- and strain-specific sensitivities to dioxins. A subregion in this domain, the Q-rich stretch, is aberrant in the hamster, and this feature may account for, or at least contribute to, the exceptional resistance of this species to the acute lethality of TCDD. The inverse correlation found between the number of glutamine residues in Q-rich subdomain and TCDD sensitivity might be relevant to human risk assessment and warrants further studies.

The influence of TCDD on expression of its own receptor in different rat strains cannot explain the wide divergences in TCDD-sensitivities among the strains. A single treatment with TCDD causes either a transient suppression of AHR expression followed by rapid recovery or a more sustained upregulation. The effect is blunted after continuous exposure.

The potent in vitro AHR agonist and antagonist, ICZ and resveratrol, respectively, show very weak AHR-related activity in rats in vivo, probably due to swift biotransformation.
 
 

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