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.     

Restructured transactivation domain in hamster AH receptor

Hamsters and Han/Wistar (Kuopio; H/W) rats show peculiarly selective responsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). They are extremely resistant to its acute lethality but sensitive to, e.g. , enzyme induction. The biological effects of TCDD are mediated by the AH receptor (AHR). Recent studies on H/W rat AHR discovered a remodelled transactivation domain which appears to be critical for the TCDD resistance of these animals. Here, molecular cloning and sequencing of hamster AHR reveals another type of restructured transactivation domain. In hamsters, the functionally pivotal Q-rich region is substantially expanded and enriched in glutamine compared with all other AHRs cloned to date. By contrast, the amino-terminal end is highly conserved, which is in agreement with the H/W rat AHR. Because of the additional material in the transactivation domain, hamster AHR protein is larger than that in rats or mice, but the pattern of AHR mRNA expression in tissues is similar.     

The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-loop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not yet been established. Moreover, no rigorous studies have been done to characterize the biochemical properties of the bHLH domain of ARNT that would address this issue. To begin this characterization, we chemically synthesized a 56-residue peptide encompassing the bHLH domain of ARNT (residues 90-145). In the absence of DNA, the ARNT-bHLH peptide can form homodimers in lower ionic strength, as evidenced by dynamic light scattering analysis, and can bind E-box DNA (CACGTG) with high specificity and affinity, as determined by fluorescence anisotropy. Dimers and tetramers of ARNT-bHLH are observed bound to DNA in equilibrium sedimentation and dynamic light scattering experiments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strongly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentrations supporting a physiological function and underscore the significant biochemical differences among the bHLH superfamily.     

Expression of the mediators of dioxin toxicity,aryl hydrocarbon receptor (AHR) and the AHR nuclear translocator (ARNT), is developmentally regulated in mouse teeth

Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.     

Molecular characterization of the aryl hydrocarbon receptors (AHR1 and AHR2) from red seabream (Pagrus major)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs). Bony fishes exposed to PHAHs exhibit a wide range of developmental defects. However, functional roles of fish AHR are not yet fully understood, compared with those of mammalian AHRs. To investigate the potential sensitivity to PHAHs toxic effects, an AHR cDNA was initially cloned and sequenced from red seabream (Pagrus major), an important fishery resource in Japan. The present study succeeded in identifying two highly divergent red seabream AHR cDNA clones, which shared only 32% identity in full-length amino acid sequence. The phylogenetic analysis revealed that one belonged to AHR1 clade (rsAHR1) and another to AHR2 clade (rsAHR2). The rsAHR1 encoded a 846-residue protein with a predicted molecular mass of 93.2 kDa, and 990 amino acids and 108.9 kDa encoded rsAHR2. In the N-terminal half, both rsAHR genes included bHLH and PAS domains, which participate in ligand binding, AHR/ARNT dimerization and DNA binding. The C-terminal half, which is responsible for transactivation, was poorly conserved between rsAHRs. Quantitative analyses of both rsAHRs mRNAs revealed that their tissue expression profiles were isoform-specific; rsAHR1 mRNA expressed primarily in brain, heart, ovary and spleen, while rsAHR2 mRNA was observed in all tissues examined, indicating distinct roles of each rsAHR. Furthermore, there appeared to be species-differences in the tissue expression profiles of AHR isoforms between red seabream and other fish. These results suggest that there are isoform- and species-specific functions in piscine AHRs.