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.

Developmental and tissue-specific expression of AHR1, AHR2, and ARNT2 in dioxin-sensitive and -resistant populations of the marine fish Fundulus heteroclitus.

Fundulus heteroclitus is a well-characterized marine fish model for studying aryl hydrocarbon toxicity. The F. heteroclitus population in New Bedford Harbor (NBH), a Superfund site in southeastern Massachusetts, exhibits heritable resistance to the toxic effects of planar halogenated aromatic hydrocarbons (PHAHs), including 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs). To investigate the role of the aryl hydrocarbon receptor (AHR) signal transduction pathway in PHAH resistance, we measured the relative levels of AHR1, AHR2, and ARNT2 mRNA in whole embryos at different developmental stages and in dissected tissues of adults, comparing expression of these genes in NBH fish with fish from a reference site (Scorton Creek, MA [SC]). Expression of both AHR1 and AHR2 mRNA increased during development, achieving maximum levels prior to hatching. Maximal embryonic expression of AHR1 was delayed relative to AHR2. Whole NBH and SC embryos exhibited no discernable differences in expression of these genes. As we have previously observed, adult SC fish expressed AHR2 and ARNT2 mRNA in all tissues examined, while AHR1 was expressed predominantly in brain, heart, and gonads. In contrast, AHR1 mRNA was widely expressed in NBH fish, appearing with unusual abundance in gill, gut, kidney, liver, and spleen. This AHR1 expression pattern was not observed in the lab-reared progeny of NBH fish, demonstrating that constitutive AHR1 expression in gill, gut, kidney, liver, and spleen is not a heritable phenotype. Furthermore, widespread AHR1 expression was not induced in reference-site fish by TCDD or PCB mixtures, suggesting that aberrant AHR1 expression is not simply a normal physiological response of contaminant exposure. These results identify ubiquitous AHR1 expression as an attribute unique to feral NBH F. heteroclitus, and they represent a first step in determining the regulatory mechanisms underlying this expression pattern and its possible role in TCDD resistance.

The evolution of aryl hydrocarbon signaling proteins: diversity of ARNT isoforms among fish species.

The aryl hydrocarbon receptor nuclear translocator (ARNT) mediates aryl hydrocarbon signaling and toxicity by dimerizing with the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that binds specific DNA elements and alters transcription of target genes. Two genes encode different forms of ARNT in rodents: ARNT1, which is widely expressed, and ARNT2, which exhibits a very restricted expression pattern. In an effort to characterize aryl hydrocarbon signaling mechanisms in fishes, we previously isolated an ARNT cDNA from Fundulus heteroclitus and discovered that this species expresses ARNT2 ubiquitously. This situation differs not only from mammals, but also from rainbow trout, which expresses a divergent ARNT gene that we hypothesized was peculiar to salmonids (rtARNTa/b). In this communication, we examine the ARNT sequences of multiple fish species, including a newly isolated cDNA from scup (Stenotomus chrysops). Our phylogenetic analysis demonstrates that zebrafish ARNT, like the Fundulus protein, is an ARNT2. Contrary to expectations, the scup ARNT is closely related to the rainbow trout protein, demonstrating that the existence of this ARNT isoform predates the divergence of salmonids from the other teleosts. Thus, different species of fish express distinct and highly conserved isoforms of ARNT. The number, type, and expression pattern of ARNT proteins may contribute to interspecies differences in aryl hydrocarbon toxicity, possibly through distinct interactions with additional PAS-family proteins.

Identification and functional characterization of two highly divergent aryl hydrocarbon receptors (AHR1 and AHR2) in the teleost Fundulus heteroclitus. Evidence for a novel subfamily of ligand-binding basic helix loop helix-Per-ARNT-Sim (bHLH-PAS) factors.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds cause altered gene expression and toxicity. The AHR belongs to an emerging multigene family of transcription factors possessing basic helix loop helix (bHLH) and Per-ARNT-Sim (PAS) domains. Most bHLH-PAS proteins occur as duplicates or "paralog groups" in mammals, but only a single mammalian AHR has been identified. Here we report the cDNA cloning of two distinct AHRs, designated FhAHR1 and FhAHR2, from a single vertebrate species, the teleost Fundulus heteroclitus (Atlantic killifish). Both Fundulus AHR proteins possess bHLH and PAS domains that are closely related to those of the mammalian AHR. FhAHR1 and FhAHR2 are highly divergent (40% overall amino acid identity; 61% identity in the N-terminal half), suggesting that they arose from a gene duplication predating the divergence of mammals and fish. Photoaffinity labeling with 2-azido-3-[(125)I]iodo-7, 8-dibromodibenzo-p-dioxin and velocity sedimentation analysis using 2,3,7,8-[1,6-(3)H]TCDD showed that both FhAHR1 and FhAHR2 exhibit specific, high-affinity binding of dioxins. Both AHRs also showed specific, TCDD- and ARNT-dependent interactions with a mammalian xenobiotic response element. The two Fundulus AHR genes displayed different tissue-specific patterns of expression; FhAHR1 transcripts were primarily expressed in brain, heart, ovary, and testis, while FhAHR2 transcripts were equally abundant in many tissues. Phylogenetic analysis demonstrated that Fundulus AHR1 is an ortholog of mammalian AHRs, while AHR2 forms in Fundulus and other fish are paralogous to Fundulus AHR1 and the mammalian AHRs and thus represent a novel vertebrate subfamily of ligand-binding AHRs.

Functional diversity of vertebrate ARNT proteins: identification of ARNT2 as the predominant form of ARNT in the marine teleost, Fundulus heteroclitus.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH/PAS protein superfamily. ARNT dimerizes with several PAS superfamily members, including the ligand-activated aryl hydrocarbon receptor (AHR), forming a complex that alters transcription by binding specific elements within the promoters of target genes. Two genes encode different forms of the protein in rodents: ARNT1, which is widely expressed, and ARNT2, which is limited to the brain and kidneys of adults and specific neural and branchial tissues of embryos. In an effort to characterize aryl hydrocarbon signaling mechanisms in Fundulus heteroclitus, a marine teleost that can develop heritable xenobiotic resistance, we have isolated a liver cDNA encoding an ARNT homolog. The protein exhibits AHR-dependent DNA binding capability typical of other vertebrate ARNTs. Unexpectedly, phylogenetic analysis reveals that the cDNA encodes an ARNT2. This is the only detectable ARNT sequence in Fundulus liver, gill, ovary, and brain, suggesting that ARNT2 is the predominant form of ARNT in this species. Also surprising is the relative lack of sequence identity with another fish ARNT protein, rainbow trout ARNTb, which we show forms a distinct branch outside the ARNT1 and ARNT2 clades in phylogenetic analyses. Functional diversity of ARNT proteins in fish may have important implications for the assessment of aryl hydrocarbon effects on natural populations. The increasing use of fish models in developmental and toxicological studies underscores the importance of identifying taxon-specific roles of ARNT proteins and their potential dimeric partners in the PAS superfamily.

Dexamethasone stimulates release of an ANP-like substance from rainbow trout cardiocytes.

A substance that cross-reacts with antiserum to human atrial natriuretic peptide (ANP) is found in fish hearts. This ANP-like material increases sodium output from the gill and kidney while inhibiting sodium uptake in the gut. Mammalian ANP secretion is stimulated by glucocorticoids, and cortisol injection increases sodium output in salt-loaded fish. Therefore, we wanted to determine if the release of ANP in fish is sensitive to dexamethasone. Ventricle cardiocytes from the rainbow trout Oncorhynchus mykiss were treated with various doses of dexamethasone for 18 or 72 h. Single ventricle cells were then assayed for ANP release using a reverse hemolytic plaque assay and antiserum to human alpha-ANP. Incubation with 100 microM dexamethasone almost doubled the population of ventricle cells committed to ANP release (basal, 15.0 +/- 0.3% vs. Dexamethasone, 28.3 +/- 1.4%; values are percent plaque formation +/- SE). Stimulation of ANP secretion was dependent on dose and time of exposure to dexamethasone. These results suggest that ANP secretion in fish is regulated by glucocorticoids.