Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.     

Molecular evidence predicts aryl hydrocarbon receptor ligand insensitivity in the peregrine falcon (<Emphasis Type="Italic">Falco peregrines</Emphasis>)

Some wild populations of fish-eating birds and raptors are exposed to high concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds such as other 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans and coplanar polychlorinated biphenyls, resulting in accumulation in their tissues. It has been demonstrated that TCDD-like chemicals cause toxic effects via aryl hydrocarbon receptor (AHR)-mediated signaling pathways. The aim of this study was to characterize the AHR from the peregrine falcon (Falco peregrines) to predict its sensitivity to TCDD-like chemicals. The AHR1, AHR2, and AHR nuclear translocator 1 of the peregrine falcon are more similar in amino acid sequence to avian species less sensitive to TCDD-like chemicals such as the cormorant (95%) than to more sensitive species such as the chicken (90%). From the amino acid sequence, it is likely that the ligand-binding affinity of peregrine falcon AHR1 and AHR2 would be very low compared with the chicken or other sensitive species, and it was actually proved by an in vitro reporter gene assay. We concluded that the peregrine falcon, one of raptor species, may be relatively resistant to TCDD-like chemicals.     

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.     

Genetic diversity at an oncogene locus and in mitochondrial DNA between populations of cancer-prone atlantic tomcod

It has been reported that Atlantic tomcod (Microgadus tomcod) from the Hudson River exhibit an extremely high incidence of liver tumors. More than 90% of spawning 2-year-old fish display hepatocellular carcinomas. In contrast, representatives of this species from a relatively pristine environment show a much lower incidence of tumors. Genomic DNA and mitochondrial DNA (mtDNA) were isolated from tomcod from the Hudson River, New York, and the Saco River and Royal River, Maine. We found a statistically significant difference in the frequency of PstI-generated restriction fragment length polymorphisms in the abl cellular oncogene between Hudson and Maine tomcod. Allelic variation was observed at two of the three abl domains scored. A single composite genotype seen in approximately 40% of Hudson River fish was seen in only one Maine fish. This polymorphism enabled us to differentiate a Hudson River population from that encountered in the Maine rivers. This is the first demonstration of a population-specific polymorphism at a cellular oncogene locus in any species. In contrast, no restriction site polymorphisms were seen in mtDNA between the populations. The lack of mtDNA diversity in these fish is consistent with the geological history of the area. In combination, these results suggest that the genetic diversity observed at the c-abl oncogene locus must have been a fairly recent event and that oncogene loci may be particularly sensitive to mutational change.     

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.     

Effects of Aroclor 1254 treatment on the in vitro hepatic metabolism of toluene, aniline and aminopyrine in hybrid sunfish

The in vitro metabolism of the volatile aromatic hydrocarbon toluene by enzymes associated with the 12,000 g supernatant fraction of hybrid sunfish (Lepomis macrochirus X L. cyanellus X L. gibbosus) liver homogenates was studied. Aminopyrine demethylase (APDM) and aniline hydroxylase (AH) activities were measured. Intramuscular injections of Aroclor 1254 (a polychlorinated biphenyl) produced significant increases in APDM and AH activities (P less than or equal to 0.1, ANOVA). There were no significant changes in the metabolism of toluene, liver wet weights, or liver protein concentrations following treatment.     

Comparative investigation of cyclophosphamide action on bone marrow cells of the Armenian hamster and 4 other species of rodents.

We studied the clastogenic action of cyclophosphamide (CP) on bone marrow cells of the Armenian hamster (AH), Cricetulus migratorius. CP induced a dose-dependent linear increase in aberrant cells. The maximal cytogenetic action was observed 12 h after CP treatment. Male and female AHs were similarly sensitive to the clastogenic action of CP. We compared CP clastogenicity at a dose of 25 mg/kg on bone marrow cells of AHs, mice, rats, guinea pigs and Chinese hamsters 24 h after treatment. We observed that this dose of CP induced only 2.8% aberrant cells in bone marrow of AHs, but 42.8%, 32.2%, 25% and 14.6% aberrant cells in bone marrow of guinea pigs, rats, mice and Chinese hamsters respectively. AHs are much more resistant to the metaphase-arresting action of colchicine than other species of rodents (e.g., the colchicine dose for AHs is 100-fold more than for rats). Thus AHs are the most resistant of all rodent species studied to the clastogenic action of CP.     

Xenobiotic metabolizing enzymes in spawning English sole (Parophrys vetulus) exposed to organic-solvent extracts of marine sediments from contaminated and reference areas

Female English sole (Parophrys vetulus) within 1-2 days of spawning were exposed by i.m. injection to organic-solvent extracts of marine sediments at the following doses: Eagle Harbor (EHSE, contaminated site)--6.8 mg aromatic hydrocarbons (AHs)/kg body wt; Duwamish Waterway (DSE, contaminated site)--0.52 mg AHs and 0.040 mg chlorinated hydrocarbons (CHs)/kg body wt; Hood Canal (HCSE, reference site)--0.00090 mg AHs/kg body wt. Hepatic aryl hydrocarbon hydroxylase (AHH) activity, measured at spawning, was induced 10-, 23-and 2-fold by EHSE, DSE and HCSE, respectively, compared to sham and vehicle controls. Hepatic glutathione-S-transferase and epoxide hydrolase activities were not affected by any treatment. EHSE, but not DSE or HCSE, inhibited spawning (P less than 0.01) in 36% of the exposed fish and hepatic AHH activity in the non-spawning fish was significantly (P less than 0.05) higher than in the fish that did spawn. These results suggest a potential for reproductive toxicity in benthic fish after exposure to sediment-associated contaminants.     

Zebrafish whole-adult-organism chemogenomics for large-scale predictive and discovery chemical biology

The ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology.     

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.     

Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon receptors (AHRs)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental contaminants that pose a potential risk to fish populations. Both field and laboratory studies suggest that exposure of the early life stages of fish to PAH can mimic the embryotoxic effects of the planar halogenated hydrocarbons (PHHs), the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin. PHH toxicity is mediated by the aryl hydrocarbon receptor (AHR) and PHH potency is predicted by its AHR-binding affinity and CYP1A induction potency. However, the role of the AHR, if any, in mediating the developmental effects of PAH to fish remains unknown. In this study we looked at the AHR binding affinity of a test set of PAH that had been previously ranked for their potency for inducing teleost CYP1A. PAH that induced CYP1A inhibited [3H]TCDD binding to in vitro-expressed AHRs from rainbow trout and the AHR expressed in PLHC-1 fish hepatoma cells. Generally, the relative rank order for AHR binding affinity predicted the rank order of these same PAH for inducing CYP1A reported in other studies. There was a strong, positive relationship between binding to the PLHC-1 AHR (stimulus) and the EC50s for CYP1A induction (response) in whole juvenile trout and in RTL-W1 cells, but EC50s were much higher than expected for a 1:1 stimulus/response relationship. These data show that the ability of PAH to bind to teleost AHR predicts PAH potency for CYP1A induction. If PAH toxicity is receptor-mediated and predicted by induction potencies, we will have a powerful mechanistic-based tool for rapidly assessing the risk of toxicity to fish of PAH from any source.