Identification of novel splice variants of ARNT and ARNT2 in the rat

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.     

The AH receptor of the most dioxin-sensitive species, guinea pig, is highly homologous to the human AH receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) brings about a wide spectrum of toxic and biochemical changes, most of which are mediated by the AH receptor (AHR). Recent cloning of the AHR from the two most TCDD-resistant laboratory animals, Han/Wistar (Kuopio) rats and hamsters, suggested a critical role for the C-terminal transactivation domain structure in TCDD sensitivity. Here we cloned the AHR from the most TCDD-susceptible species, guinea pig. The N-terminus of its AHR was highly similar to that in the resistant animals. However, the C-terminal Q-rich subdomain was only about half the size of this subunit in the hamster AHR. There was a distinct correlation across published mammalian species between the number of glutamine residues in the Q-rich subdomain and sensitivity to the acute lethality of TCDD. The closest homolog of the Guinea pig receptor turned out to be the human AHR, which may be relevant for dioxin risk assessment.     

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.     

A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity

The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.     

Polymorphisms in the human AH receptor

The AH receptor (AHR) mediates toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as well as induction of three cytochrome P450 enzymes and certain Phase II enzymes. In laboratory animals, genetic variations in the AHR lead to substantial differences in sensitivity to biochemical and toxic effects of TCDD and related compounds. Relatively few polymorphisms have been discovered in the human AHR gene; these occur predominantly in exon 10, a region that encodes a major portion of the transactivation domain of the receptor that is responsible for regulating expression of other genes. In human populations there is a wide range of variation in responses regulated by the AHR for example, induction of CYP1A1. Some variation in human responsiveness likely is due to genetically based variations in AHR structure. Thus far, however, only one pair of polymorphisms, those at codons 517 and 570, has been shown to have a clear cut and strong effect on the phenotype of an AHR-mediated response. The search continues for polymorphisms that alter AHR function because this receptor is a central factor in determining responses to important environmental contaminants and also plays a physiologic role in early development in mammals.     

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.     

An aryl hydrocarbon receptor (AHR) homologue from the soft-shell clam, Mya arenaria: evidence that invertebrate AHR homologues lack 2,3,7,8-tetrachlorodibenzo-p-dioxin and beta-naphthoflavone binding.

The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.     

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome p450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome p450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-beta) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.