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Saxitoxin (STX) is a potent neurotoxin naturally produced by dinoflagellates and cyanobacteria. STX inhibits voltage-gated sodium channels (VGSCs), affecting the propagation of action potentials. Consumption of seafood contaminated with STX is responsible for paralytic shellfish poisoning (PSP). Humans are among the species most sensitive to PSP; neurological symptoms of exposure range from tingling of the extremities to severe paralysis. The objective of this study was to determine the effects of STX exposure on developmental processes during early embryogenesis. This study was designed to test the hypothesis that early developmental exposure to STX would disrupt key processes, particularly those related to neural development. Zebrafish embryos were exposed to STX (24 or 48 pg) or vehicle (0.3 mM HCl) at 6 h post fertilization (hpf) via microinjection. There was no overt toxicity but starting at 36 hpf there was a temporary lack of pigmentation in STX-injected embryos, which resolved by 72 hpf. Using high performance liquid chromatography, we found that STX was retained in embryos up to 72 hpf in a dose-dependent manner. Temporal transcriptional profiling of embryos exposed to 48 pg STX per embryo revealed no differentially expressed genes (DEGs) at 24 hpf, but at 36 and 48 hpf, there were 3547 and 3356 DEGs, respectively. KEGG pathway analysis revealed significant enrichment of genes related to focal adhesion, adherens junction and regulation of actin cytoskeleton, suggesting that cell-cell and cell-extracellular matrix interactions were affected by STX. Genes affected are critical for axonal growth and the development of functional neural networks. We confirmed these findings by visualizing axonal defects in transgenic zebrafish with fluorescently labeled sensory neurons. In addition, our gene expression results suggest that STX exposure affects both canonical and noncanonical functions of VGSCs. Given the fundamental role of VGSCs in both physiology and development, these findings offer valuable insights into effects of exposure to neurotoxins., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The genetic architecture of phenotypic traits can affect the mode and tempo of trait evolution. Human-altered environments can impose strong natural selection, where successful evolutionary adaptation requires swift and large phenotypic shifts. In these scenarios, theory predicts that adaptation is due to a few adaptive variants of large effect, but empirical studies that have revealed the genetic architecture of rapidly evolved phenotypes are rare, especially for populations inhabiting polluted environments. Fundulus killifish have repeatedly evolved adaptive resistance to extreme pollution in urban estuaries. Prior studies, including genome scans for signatures of natural selection, have revealed some of the genes and pathways important for evolved pollution resistance, and provide context for the genotype-phenotype association studies reported here. We created multiple quantitative trait locus (QTL) mapping families using progenitors from four different resistant populations, and using RAD-seq genetically mapped variation in sensitivity (developmental perturbations) following embryonic exposure to a model toxicant PCB-126. We found that one to two large-effect QTL loci accounted for resistance to PCB-mediated developmental toxicity. QTLs harbored candidate genes that govern the regulation of aryl hydrocarbon receptor (AHR) signaling. One QTL locus was shared across all populations and another was shared across three populations. One QTL locus showed strong signatures of recent natural selection in the corresponding wild population but another QTL locus did not. Some candidate genes for PCB resistance inferred from genome scans in wild populations were identified as QTL, but some key candidate genes were not. We conclude that rapidly evolved resistance to the developmental defects normally caused by PCB-126 is governed by few genes of large effect. However, other aspects of resistance beyond developmental phenotypes may be governed by additional loci, such that comprehensive resistance to PCB-126, and to the mixtures of chemicals that distinguish urban estuaries more broadly, may be more genetically complex., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Sunlight transforms plastic into water-soluble products, the potential toxicity of which remains unresolved, particularly for vertebrate animals. We evaluated acute toxicity and gene expression in developing zebrafish larvae after 5 days of exposure to photoproduced (P) and dark (D) leachates from additive-free polyethylene (PE) film and consumer-grade, additive-containing, conventional, and recycled PE bags. Using a "worst-case" scenario, with plastic concentrations exceeding those found in natural waters, we observed no acute toxicity. However, at the molecular level, RNA sequencing revealed differences in the number of differentially expressed genes (DEGs) for each leachate treatment: thousands of genes (5442 P, 577 D) for the additive-free film, tens of genes for the additive-containing conventional bag (14 P, 7 D), and none for the additive-containing recycled bag. Gene ontology enrichment analyses suggested that the additive-free PE leachates disrupted neuromuscular processes via biophysical signaling; this was most pronounced for the photoproduced leachates. We suggest that the fewer DEGs elicited by the leachates from conventional PE bags (and none from recycled bags) could be due to differences in photoproduced leachate composition caused by titanium dioxide-catalyzed reactions not present in the additive-free PE. This work demonstrates that the potential toxicity of plastic photoproducts can be product formulation-specific.

Crude oil released into the environment undergoes weathering processes that gradually change its composition and toxicity. Co-exposure to petroleum mixtures and other stressors, including ultraviolet (UV) radiation, may lead to synergistic effects and increased toxicity. Laboratory studies should consider these factors when testing the effects of oil exposure on aquatic organisms. Here, we study transcriptomic responses of the estuarine sea anemone Nematostella vectensis to naturally weathered oil, with or without co-exposure to environmental levels of UV radiation. We find that co-exposure greatly enhances the response. We use bioinformatic analyses to identify molecular pathways implicated in this response, which suggest phototoxicity and oxidative damage as mechanisms for the enhanced stress response. Nematostella's stress response shares similarities with the vertebrate oxidative stress response, implying deep conservation of certain stress pathways in animals. We show that exposure to weathered oil along with surface-level UV exposure has substantial physiological consequences in a model cnidarian., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3' UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate 5%), respectively. The majority of the peaks were preferentially located around the 3' UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor that mediates the toxicity of halogenated and polycyclic aromatic hydrocarbons in vertebrates. Atlantic cod ( Gadus morhua ) has recently emerged as a model organism in environmental toxicology studies, and increased knowledge of Ahr-mediated responses to xenobiotics is imperative. Genome mining and phylogenetic analyses revealed two Ahr-encoding genes in the Atlantic cod genome, gmahr1a and gmahr2a . In vitro binding assays showed that both gmAhr proteins bind to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), but stronger binding to gmAhr1a was observed. Transactivation studies with a reporter gene assay revealed that gmAhr1a is one order of magnitude more sensitive to TCDD than gmAhr2a, but the maximal responses of the receptors were similar. Other well-known Ahr agonists, such as β-naphthoflavone (BNF), 3,3',4,4',5-pentachlorobiphenyl (PCB126), and 6-formylindolo[3,2- b ]carbazole (FICZ), also activated the gmAhr proteins, but gmAhr1a was, in general, the more sensitive receptor and produced the highest efficacies. The induction of cyp1a in exposed precision-cut cod liver slices confirmed the activation of the Ahr signaling pathway ex vivo. In conclusion, the differences in transcriptional activation by gmAhr's with various agonists, the distinct binding properties with TCDD and BNF, and the distinct tissue-specific expression profiles indicate different functional specializations of the Atlantic cod Ahr's.

Polychlorinated biphenyls (PCBs) are highly persistent and ubiquitously distributed environmental pollutants. Based on their chemical structure, PCBs are classified into non-ortho-substituted and ortho-substituted congeners. Non-ortho-substituted PCBs are structurally similar to dioxin and their toxic effects and mode of action are well-established. In contrast, very little is known about the effects of ortho-substituted PCBs, particularly, during early development. The objective of this study is to investigate the effects of exposure to an environmentally prominent ortho-substituted PCB (2,2',4,4',5,5'-hexachlorobiphenyl; PCB153) on zebrafish embryos. We exposed zebrafish embryos to 3 different concentrations of PCB153 starting from 4 to 120 hours post-fertilization (hpf). We quantified gross morphological changes, behavioral phenotypes, gene expression changes, and circadian behavior in the larvae. There were no developmental defects during the exposure period, but starting at 7 dpf, we observed spinal deformity in the 10 μM PCB153 treated group. A total of 633, 2227, and 3378 differentially expressed genes were observed in 0.1 μM (0.036 μg/ml), 1 μM (0.36 μg/ml), and 10 μM (3.6 μg/ml) PCB153-treated embryos, respectively. Of these, 301 genes were common to all treatment groups. KEGG pathway analysis revealed enrichment of genes related to circadian rhythm, FoxO signaling, and insulin resistance pathways. Behavioral analysis revealed that PCB153 exposure significantly alters circadian behavior. Disruption of circadian rhythms has been associated with the development of metabolic and neurological diseases. Thus, understanding the mechanisms of action of environmental chemicals in disrupting metabolism and other physiological processes is essential., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100 nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sentinel species such as the Atlantic killifish (Fundulus heteroclitus) living in urban waterways can be used as toxicological models to understand impacts of environmental metabolism disrupting compound (MDC) exposure on both wildlife and humans. Exposure to MDCs is associated with increased risk of metabolic syndrome, including impaired lipid and glucose homeostasis, adipogenesis, appetite control, and basal metabolism. MDCs are ubiquitous in the environment, including in aquatic environments. New Bedford Harbor (NBH), Massachusetts is polluted with polychlorinated biphenyls (PCBs), and, as we show for the first time, tin (Sn). PCBs and organotins are ligands for two receptor systems known to regulate lipid homeostasis, the aryl hydrocarbon receptor (AHR) and the peroxisome proliferator-activated receptors (PPARs), respectively. In the current study, we compared lipid homeostasis in laboratory-reared killifish from NBH (F2) and a reference location (Scorton Creek, Massachusetts; F1 and F2) to evaluate how adaptation to local conditions may influence responses to MDCs. Adult killifish from each population were exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB126, dioxin-like), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153, non-dioxin-like), or tributyltin (TBT, a PPARγ ligand) by a single intraperitoneal injection and analyzed after 3 days. AHR activation was assessed by measuring cyp1a mRNA expression. Lipid homeostasis was evaluated phenotypically by measuring liver triglycerides and organosomatic indices, and at the molecular level by measuring the mRNA expression of pparg and ppara and a target gene for each receptor. Acute MDC exposure did not affect phenotypic outcomes. However, overall NBH killifish had higher liver triglycerides and adiposomatic indices than SC killifish. Both season and population were significant predictors of the lipid phenotype. Acute MDC exposure altered hepatic gene expression only in male killifish from SC. PCB126 exposure induced cyp1a and pparg, whereas PCB153 exposure induced ppara. TBT exposure did not induce ppar-dependent pathways. Comparison of lipid homeostasis in two killifish populations extends our understanding of how MDCs act on fish and provides a basis to infer adaptive benefits of these differences in the wild., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Interactions between regulatory pathways allow organisms to adapt to their environment and respond to stress. One interaction that has been recently identified occurs between the aryl hydrocarbon receptor (AHR) and the nuclear factor erythroid-2 related factor (NRF) family. Each transcription factor regulates numerous downstream genes involved in the cellular response to toxicants and oxidative stress; they are also implicated in normal developmental pathways. The zebrafish model was used to explore the role of AHR regulation of nrf genes during development and in response to toxicant exposure. To determine if AHR1b is responsible for transcriptional regulation of 6 nrf genes during development, a loss-of-function experiment using morpholino-modified oligonucleotides was conducted followed by a chromatin immunoprecipitation study at the beginning of the pharyngula period (24 h postfertilization). The expression of nrf1a was AHR1b dependent and its expression was directly regulated through specific XREs in its cis-promoter. However, nrf1a expression was not altered by exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a toxicant and prototypic AHR agonist. The expression of nrf1b, nrf2a, and nfe2 was induced by TCDD, and AHR1b directly regulated their expression by binding to cis-XRE promoter elements. Last, nrf2b and nrf3 were neither induced by TCDD nor regulated by AHR1b. These results show that AHR1b transcriptionally regulates nrf genes under toxicant modulation via binding to specific XREs. These data provide a better understanding of how combinatorial molecular signaling potentially protects embryos from embryotoxic events following toxicant exposure.

There is growing evidence that environmental toxicants can affect various physiological processes by altering DNA methylation patterns. However, very little is known about the impact of toxicant-induced DNA methylation changes on gene expression patterns. The objective of this study was to determine the genome-wide changes in DNA methylation concomitant with altered gene expression patterns in response to 3, 3', 4, 4', 5-pentachlorobiphenyl (PCB126) exposure. We used PCB126 as a model environmental chemical because the mechanism of action is well-characterized, involving activation of aryl hydrocarbon receptor, a ligand-activated transcription factor. Adult zebrafish were exposed to 10 nM PCB126 for 24 h (water-borne exposure) and brain and liver tissues were sampled at 7 days post-exposure in order to capture both primary and secondary changes in DNA methylation and gene expression. We used enhanced Reduced Representation Bisulfite Sequencing and RNAseq to quantify DNA methylation and gene expression, respectively. Enhanced reduced representation bisulfite sequencing analysis revealed 573 and 481 differentially methylated regions in the liver and brain, respectively. Most of the differentially methylated regions are located more than 10 kilobases upstream of transcriptional start sites of the nearest neighboring genes. Gene Ontology analysis of these genes showed that they belong to diverse physiological pathways including development, metabolic processes and regeneration. RNAseq results revealed differential expression of genes related to xenobiotic metabolism, oxidative stress and energy metabolism in response to polychlorinated biphenyl exposure. There was very little correlation between differentially methylated regions and differentially expressed genes suggesting that the relationship between methylation and gene expression is dynamic and complex, involving multiple layers of regulation.

Limited knowledge of the molecular evolution of deep-sea fish proteomes so far suggests that a few widespread residue substitutions in cytosolic proteins binding hydrophilic ligands contribute to resistance to the effects of high hydrostatic pressure (HP). Structure-function studies with additional protein systems, including membrane bound proteins, are essential to provide a more general picture of adaptation in these extremophiles. We explored molecular features of HP adaptation in proteins binding hydrophobic ligands, either in lipid bilayers (cytochrome P450 1A - CYP1A) or in the cytosol (the aryl hydrocarbon receptor - AHR), and their partners P450 oxidoreductase (POR) and AHR nuclear translocator (ARNT), respectively. Cloning studies identified the full-length coding sequence of AHR, CYP1A and POR, and a partial sequence of ARNT from Coryphaenoides armatus, an abyssal gadiform fish thriving down to 5000m depth. Inferred protein sequences were aligned with many non-deep-sea homologs to identify unique amino acid substitutions of possible relevance in HP adaptation. Positionally unique substitutions of various physicochemical properties were found in all four proteins, usually at sites of strong-to-absolute residue conservation. Some were in domains deemed important for protein-protein interaction or ligand binding. In addition, some involved removal or addition of beta-branched residues; local modifications of beta-branched residue patterns could be important to HP adaptation. In silico predictions further suggested that some unique substitutions might substantially modulate the flexibility of the polypeptide segment in which they are found. Repetitive motifs unique to the abyssal fish AHR were predicted to be rich in glycosylation sites, suggesting that post-translational changes could be involved in adaptation as well. Recombinant CYP1A and AHR showed functional properties (spectral characteristics, catalytic activity and ligand binding) that demonstrate proper folding at 1atm, indicating that they could be used as deep-sea fish protein models to further evaluate protein function under pressure. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone"., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, nonembryotoxic levels of 3,3',4,4',5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Non-dioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine receptors (RyR), microsomal Ca 2+ channels of broad significance. Teleost fish may be important models for NDL PCB neurotoxicity, and we used sequencing databases to characterize teleost RyR and FK506 binding protein 12 or 12.6kDa (genes FKBP1A; FKBP1B), which promote NDL PCB-triggered Ca 2+ dysregulation. Particular focus was placed on describing genes in the Atlantic killifish (Fundulus heteroclitus) genome and searching available RNA-sequencing datasets for single nucleotide variants (SNV) between PCB tolerant killifish from New Bedford Harbor (NBH) versus sensitive killifish from Scorton Creek (SC), MA. Consistent with the teleost whole genome duplication (tWGD), killifish have six RyR genes, corresponding to a and b paralogs of mammalian RyR1, 2 and 3. The presence of six RyR genes was consistent in all teleosts investigated including zebrafish. Killifish have four FKBP1; one FKBP1b and three FKBP1a named FKBP1aa, FKBP1ab, likely from the tWGD and a single gene duplicate FKBP1a3 suggested to have arisen in Atherinomorphae. The RyR and FKBP1 genes displayed tissue and developmental stage-specific mRNA expression, and the previously uncharacterized RyR3, herein named RyR3b, and all FKBP1 genes were prominent in brain. We identified a SNV in RyR3b encoding missense mutation E1458D. In NBH killifish, 57% were heterozygous and 28% were homozygous for this SNV, whereas almost all SC killifish (94%) lacked the variant (n≥39 per population). The outlined sequence differences between mammalian and teleost RyR and FKBP1 together with outlined population differences in SNV frequency may contribute to our understanding of NDL PCB neurotoxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The hypoxia-inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus is found in habitats prone to hypoxia. It responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, HIF2α, and HIF3α independently formed complexes with the β-subunit, aryl hydrocarbon receptor nuclear translocator, to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α-HIF2αb-a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations., (Copyright © 2017 the American Physiological Society.)

The aryl hydrocarbon receptor (AHR) was for many years of interest only to pharmacologists and toxicologists. However, this protein has fundamental roles in biology that are being revealed through studies in diverse animal species. The AHR is an ancient protein. AHR homologs exist in most major groups of modern bilaterian animals, including deuterostomes (chordates, hemichordates, echinoderms) and the two major clades of protostome invertebrates [ecdysozoans (e.g. arthropods and nematodes) and lophotrochozoans (e.g. molluscs and annelids)]. AHR homologs also have been identified in cnidarians such as the sea anemone Nematostella and in the genome of Trichoplax , a placozoan. Bilaterians, cnidarians, and placozoans form the clade Eumetazoa , whose last common ancestor lived approximately 600 million years ago (MYA). The presence of AHR homologs in modern representatives of all these groups indicates that the original eumetazoan animal possessed an AHR homolog. Studies in invertebrates and vertebrates reveal parallel functions of AHR in the development and function of sensory neural systems, suggesting that these may be ancestral roles. Vertebrate animals are characterized by the expansion and diversification of AHRs, via gene and genome duplications, from the ancestral protoAHR into at least five classes of AHR-like proteins: AHR, AHR1, AHR2, AHR3, and AHRR. The evolution of multiple AHRs in vertebrates coincided with the acquisition of high-affinity binding of halogenated and polynuclear aromatic hydrocarbons and the emergence of adaptive functions involving regulation of xenobiotic-metabolizing enzymes and roles in adaptive immunity. The existence of multiple AHRs may have facilitated subfunction partitioning and specialization of specific AHR types in some taxa. Additional research in diverse model and non-model species will continue to enrich our understanding of AHR and its pleiotropic roles in biology and toxicology.

Atlantic killifish populations have rapidly adapted to normally lethal levels of pollution in four urban estuaries. Through analysis of 384 whole killifish genome sequences and comparative transcriptomics in four pairs of sensitive and tolerant populations, we identify the aryl hydrocarbon receptor-based signaling pathway as a shared target of selection. This suggests evolutionary constraint on adaptive solutions to complex toxicant mixtures at each site. However, distinct molecular variants apparently contribute to adaptive pathway modification among tolerant populations. Selection also targets other toxicity-mediating genes and genes of connected signaling pathways; this indicates complex tolerance phenotypes and potentially compensatory adaptations. Molecular changes are consistent with selection on standing genetic variation. In killifish, high nucleotide diversity has likely been a crucial substrate for selective sweeps to propel rapid adaptation., (Copyright © 2016, American Association for the Advancement of Science.)

Common and roseate terns are migratory piscivorous seabirds with major breeding colonies within feeding range of the polychlorinated biphenyl (PCB)-contaminated New Bedford Harbor (NBH, MA, USA) Superfund site. Our longitudinal study shows that before PCB discharges into NBH ceased (late 1970s), tern eggs had very high but variable PCB concentrations. However, egg concentrations of PCBs as well as DDE (1,1-bis(p-chlorophenyl)-2,2-dichloroethene), the degradation product of the ubiquitous global contaminant DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane), have since declined. Rate constants for temporal decline of PCB congeners in tern eggs varied inversely with log10KOW (n-octanol-water partition coefficient), shifting egg congener patterns away from those characterizing NBH sediment. To estimate the toxic effects on tern eggs of PCB dioxin-like congener (DLC) exposures, we extrapolated published laboratory data on common terns to roseate terns by characterizing genetic and functional similarities in species aryl hydrocarbon receptors (AHRs), which mediate DLC sensitivity. Our assessment of contaminant risks suggests that terns breeding near NBH were exposed historically to toxic levels of PCBs and DDE; however, acute effects on tern egg development have become less likely since the 1970s. Our approach demonstrates how comparative genetics at target loci can effectively increase the range of inference for chemical risk assessments from tested to untested and untestable species.

Oxidative stress is an important mechanism of chemical toxicity, contributing to developmental toxicity and teratogenesis as well as to cardiovascular and neurodegenerative diseases and diabetic embryopathy. Developing animals are especially sensitive to effects of chemicals that disrupt the balance of processes generating reactive species and oxidative stress, and those anti-oxidant defenses that protect against oxidative stress. The expression and inducibility of anti-oxidant defenses through activation of NFE2-related factor 2 (Nrf2) and related proteins is an essential process affecting the susceptibility to oxidants, but the complex interactions of Nrf2 in determining embryonic response to oxidants and oxidative stress are only beginning to be understood. The zebrafish (Danio rerio) is an established model in developmental biology and now also in developmental toxicology and redox signaling. Here we review the regulation of genes involved in protection against oxidative stress in developing vertebrates, with a focus on Nrf2 and related cap'n'collar (CNC)-basic-leucine zipper (bZIP) transcription factors. Vertebrate animals including zebrafish share Nfe2, Nrf1, Nrf2, and Nrf3 as well as a core set of genes that respond to oxidative stress, contributing to the value of zebrafish as a model system with which to investigate the mechanisms involved in regulation of redox signaling and the response to oxidative stress during embryolarval development. Moreover, studies in zebrafish have revealed nrf and keap1 gene duplications that provide an opportunity to dissect multiple functions of vertebrate NRF genes, including multiple sensing mechanisms involved in chemical-specific effects., (Copyright © 2015. Published by Elsevier Inc.)

DNA methylation is one of the most important epigenetic modifications involved in the regulation of gene expression. The DNA methylation reaction is catalyzed by DNA methyltransferases (DNMTs). Recent studies have demonstrated that toxicants can affect normal development by altering DNA methylation patterns, but the mechanisms of action are poorly understood. Hence, we tested the hypothesis that developmental exposure to TCDD affects dnmt gene expression patterns. Zebrafish embryos were exposed to 5nM TCDD for 1h from 4 to 5h post-fertilization (hpf) and sampled at 12, 24, 48, 72, and 96 hpf to determine dnmt gene expression and DNA methylation patterns. We performed a detailed analysis of zebrafish dnmt gene expression during development and in adult tissues. Our results demonstrate that dnmt3b genes are highly expressed in early stages of development, and dnmt3a genes are more abundant in later stages. TCDD exposure upregulated dnmt1 and dnmt3b2 expression, whereas dnmt3a1, 3b1, and 3b4 are downregulated following exposure. We did not observe any TCDD-induced differences in global methylation or hydroxymethylation levels, but the promoter methylation of aryl hydrocarbon receptor (AHR) target genes was altered. In TCDD-exposed embryos, AHR repressor a (ahrra) and c-fos promoters were differentially methylated. To characterize the TCDD effects on DNMTs, we cloned the dnmt promoters with xenobiotic response elements and conducted AHR transactivation assays using a luciferase reporter system. Our results suggest that ahr2 can regulate dnmt3a1, dnmt3a2, and dnmt3b2 expression. Overall, we demonstrate that developmental exposure to TCDD alters dnmt expression and DNA methylation patterns., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Killifish survive and reproduce in the New Bedford Harbor (NBH) in Massachusetts (MA), USA, a site severely contaminated with polychlorinated biphenyls (PCBs) for decades. Levels of 22 different PCB congeners were analyzed in liver from killifish collected in 2008. Concentrations of dioxin-like PCBs in liver of NBH killifish were ∼400 times higher, and the levels of non-dioxin-like PCBs ∼3000 times higher than in killifish from a reference site, Scorton Creek (SC), MA. The NBH killifish are known to be resistant to the toxicity of dioxin-like compounds and to have a reduced aryl hydrocarbon receptor (AhR) signaling response. Little is known about the responses of these fish to non-dioxin-like PCBs, which are at extraordinarily high levels in NBH fish. In mammals, some non-dioxin-like PCB congeners act through nuclear receptor 1I2, the pregnane-X-receptor (PXR). To explore this pathway in killifish, a PXR cDNA was sequenced and its molecular phylogenetic relationship to other vertebrate PXRs was determined. Killifish were also collected in 2009 from NBH and SC, and after four months in the laboratory they were injected with a single dose of either the dioxin-like PCB 126 (an AhR agonist) or the non-dioxin-like PCB 153 (a mammalian PXR agonist). Gills and liver were sampled three days after injection and transcript levels of genes encoding PXR, cytochrome P450 3A (CYP3A), P-glycoprotein (Pgp), AhR2 and cytochrome P450 1A (CYP1A) were measured by quantitative PCR. As expected, there was little effect of PCB exposure on mRNA expression of AhR2 or CYP1A in liver and gills of NBH fish. In NBH fish, but not in SC fish, there was increased mRNA expression of hepatic PXR, CYP3A and Pgp upon exposure to either of the two PCB congeners. However, basal PXR and Pgp mRNA levels in liver of NBH fish were significantly lower than in SC fish. A different pattern was seen in gills, where there were no differences in basal mRNA expression of these genes between the two populations. In SC fish, but not in NBH fish, there was increased mRNA expression of branchial PXR and CYP3A upon exposure to PCB126 and of CYP3A upon exposure to PCB153. The results suggest a difference between the two populations in non-AhR transcription factor signaling in liver and gills, and that this could involve killifish PXR. It also implies possible cross-regulatory interactions between that factor (presumably PXR) and AhR2 in liver of these fish., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluation of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuable non-traditional model, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis and to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemically mediated oxidative stress and how it may vary during development, zebrafish embryos, eleutheroembryos, or larvae at 1, 2, 3, 4, 5, and 6 days post fertilization (dpf) were exposed to DMSO (0.1%), tert-butylhydroquinone (tBHQ; 10 µM) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 2 nM) for 6 hr. Transcript abundance was assessed by real-time qRT-PCR and microarray. qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. Microarray analysis showed that 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated eleutheroembryos at 4 dpf. There was substantial overlap between genes induced in developing zebrafish and a set of marker genes induced by oxidative stress in mammals. Genes induced by tBHQ in 4-dpf zebrafish included those involved in glutathione synthesis and utilization, signal transduction, and DNA damage/stress response. The strong induction of hsp70 determined by microarray was confirmed by qRT-PCR and by use of transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under control of the hsp70 promoter. Genes strongly down-regulated by tBHQ included mitfa, providing a molecular explanation for the loss of pigmentation in tBHQ-exposed embryos. These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, that responsiveness varies with development in a gene-specific manner, and that the oxidative stress response is substantially conserved in vertebrate animals.

The aryl hydrocarbon receptor (AHR) is a member of the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family of transcription factors and has diverse roles in development, physiology, and environmental sensing in bilaterian animals. Studying the expression of conserved genes and function of proteins in outgroups to protostomes and deuterostomes assists in understanding the antiquity of gene function and deciphering lineage-specific differences in these bilaterian clades. We describe the developmental expression of AHR from the sea anemone Nematostella vectensis and compare its expression with three other members of the bHLH-PAS family (AHR nuclear translocator (ARNT), Cycle, and a proto-Single-Minded/Trachealess). NvAHR expression was highest early in the larval stage with spatial expression in the basal portion of the ectoderm that became increasingly restricted to the oral pole with concentrated expression in tentacles of the juvenile polyp. The other bHLH-PAS genes showed a divergent expression pattern in later larval stages and polyps, in which gene expression was concentrated in the aboral end, with broader expression in the endoderm later in development. In co-immunoprecipitation assays, we found no evidence for heterodimerization of AHR with ARNT, contrary to the conservation of this specific interaction in all bilaterians studied to date. Similar to results with other invertebrate AHRs but in contrast to vertebrate AHRs, NvAHR failed to bind two prototypical xenobiotic AHR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, β-naphthoflavone). Together, our data suggest that AHR's original function in Eumetazoa likely involved developmental patterning, potentially of neural tissue. The role of heterodimerization in the function of AHR may have arisen after the cnidarian-bilaterian ancestor. The absence of xenobiotic binding to NvAHR further supports a hypothesis for a derived role of this protein in chemical sensing within the chordates.

Background: The non-migratory killifish Fundulus heteroclitus inhabits clean and polluted environments interspersed throughout its range along the Atlantic coast of North America. Several populations of this species have successfully adapted to environments contaminated with toxic aromatic hydrocarbon pollutants such as polychlorinated biphenyls (PCBs). Previous studies suggest that the mechanism of resistance to these and other "dioxin-like compounds" (DLCs) may involve reduced signaling through the aryl hydrocarbon receptor (AHR) pathway. Here we investigated gene diversity and evidence for positive selection at three AHR-related loci (AHR1, AHR2, AHRR) in F. heteroclitus by comparing alleles from seven locations ranging over 600 km along the northeastern US, including extremely polluted and reference estuaries, with a focus on New Bedford Harbor (MA, USA), a PCB Superfund site, and nearby reference sites., Results: We identified 98 single nucleotide polymorphisms within three AHR-related loci among all populations, including synonymous and nonsynonymous substitutions. Haplotype distributions were spatially segregated and F-statistics suggested strong population genetic structure at these loci, consistent with previous studies showing strong population genetic structure at other F. heteroclitus loci. Genetic diversity at these three loci was not significantly different in contaminated sites as compared to reference sites. However, for AHR2 the New Bedford Harbor population had significant FST values in comparison to the nearest reference populations. Tests for positive selection revealed ten nonsynonymous polymorphisms in AHR1 and four in AHR2. Four nonsynonymous SNPs in AHR1 and three in AHR2 showed large differences in base frequency between New Bedford Harbor and its reference site. Tests for isolation-by-distance revealed evidence for non-neutral change at the AHR2 locus., Conclusion: Together, these data suggest that F. heteroclitus populations in reference and polluted sites have similar genetic diversity, providing no evidence for strong genetic bottlenecks for populations in polluted locations. However, the data provide evidence for genetic differentiation among sites, selection at specific nucleotides in AHR1 and AHR2, and specific AHR2 SNPs and haplotypes that are associated with the PCB-resistant phenotype in the New Bedford Harbor population. The results suggest that AHRs, and especially AHR2, may be important, recurring targets for selection in local adaptation to dioxin-like aromatic hydrocarbon contaminants.

The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain (DBD) was highly conserved, with 74% identity to human PXR-DBD, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or constitutive androstane receptor (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the biological and toxic effects of a wide variety of structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While significant interspecies differences in AHR ligand binding specificity, selectivity, and response have been observed, the structural determinants responsible for those differences have not been determined, and homology models of the AHR ligand-binding domain (LBD) are available for only a few species. Here we describe the development and comparative analysis of homology models of the LBD of 16 AHRs from 12 mammalian and nonmammalian species and identify the specific residues contained within their ligand binding cavities. The ligand-binding cavity of the fish AHR exhibits differences from those of mammalian and avian AHRs, suggesting a slightly different TCDD binding mode. Comparison of the internal cavity in the LBD model of zebrafish (zf) AHR2, which binds TCDD with high affinity, to that of zfAHR1a, which does not bind TCDD, revealed that the latter has a dramatically shortened binding cavity due to the side chains of three residues (Tyr296, Thr386, and His388) that reduce the amount of internal space available to TCDD. Mutagenesis of two of these residues in zfAHR1a to those present in zfAHR2 (Y296H and T386A) restored the ability of zfAHR1a to bind TCDD and to exhibit TCDD-dependent binding to DNA. These results demonstrate the importance of these two amino acids and highlight the predictive potential of comparative analysis of homology models from diverse species. The availability of these AHR LBD homology models will facilitate in-depth comparative studies of AHR ligand binding and ligand-dependent AHR activation and provide a novel avenue for examining species-specific differences in AHR responsiveness.

The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC(50) values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied, and differences at amino acid sites 256, 257, 297, 324, 337, and 380 were identified. Site-directed mutagenesis, the LRG assay, and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of the 86 avian species to DLCs and (2) in vitro luciferase activity of AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r (2) = 0.93, p < 0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.

There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324&#95;Ser380 vs common tern--250-fold less sensitive than chicken; Val325&#95;Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324&#95;Ala380 and Val324&#95;Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324&#95;Ala380) and Japanese quail (Coturnix japonica; Val324&#95;Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.

NF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligonucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with subfunction partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.

The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knockdown via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e., phenotypic anchoring). In this chapter, we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use.

Background: Populations of Atlantic killifish (Fundulus heteroclitus) have evolved resistance to the embryotoxic effects of polychlorinated biphenyls (PCBs) and other halogenated and nonhalogenated aromatic hydrocarbons that act through an aryl hydrocarbon receptor (AHR)-dependent signaling pathway. The resistance is accompanied by reduced sensitivity to induction of cytochrome P450 1A (CYP1A), a widely used biomarker of aromatic hydrocarbon exposure and effect, but whether the reduced sensitivity is specific to CYP1A or reflects a genome-wide reduction in responsiveness to all AHR-mediated changes in gene expression is unknown. We compared gene expression profiles and the response to 3,3',4,4',5-pentachlorobiphenyl (PCB-126) exposure in embryos (5 and 10 dpf) and larvae (15 dpf) from F. heteroclitus populations inhabiting the New Bedford Harbor, Massachusetts (NBH) Superfund site (PCB-resistant) and a reference site, Scorton Creek, Massachusetts (SC; PCB-sensitive)., Results: Analysis using a 7,000-gene cDNA array revealed striking differences in responsiveness to PCB-126 between the populations; the differences occur at all three stages examined. There was a sizeable set of PCB-responsive genes in the sensitive SC population, a much smaller set of PCB-responsive genes in NBH fish, and few similarities in PCB-responsive genes between the two populations. Most of the array results were confirmed, and additional PCB-regulated genes identified, by RNA-Seq (deep pyrosequencing)., Conclusions: The results suggest that NBH fish possess a gene regulatory defect that is not specific to one target gene such as CYP1A but rather lies in a regulatory pathway that controls the transcriptional response of multiple genes to PCB exposure. The results are consistent with genome-wide disruption of AHR-dependent signaling in NBH fish.

Atlantic killifish (Fundulus heteroclitus) inhabiting the PCB-contaminated Superfund site in New Bedford Harbor (MA, USA) have evolved genetic resistance to the toxic effects of these compounds. They also lack induction of cytochrome P4501A (CYP1A) and other aryl hydrocarbon receptor (AHR)-dependent responses after exposure to AHR agonists, suggesting an overall down-regulation of the AHR signaling pathway. In this study, we hypothesized that the genetic resistance is due to altered AHR expression resulting from hypermethylation of DNA in the promoter region of AHR genes in fish inhabiting New Bedford Harbor. To test this hypothesis, we cloned and sequenced AHR1 and AHR2 promoter regions and employed bisulfite conversion-polymerase chain reaction (BS-PCR) followed by clonal analysis to compare the methylation status of CpG islands of AHR1 and AHR2 in livers of adult killifish collected from New Bedford Harbor and a reference site (Scorton Creek, MA). No significant differences in methylation profiles were observed in either AHR1 or AHR2 promoter regions between NBH and SC fish. However, hypermethylation of the AHR1 promoter correlated with low expression of transcripts in the liver in both populations. In comparison to AHR1, hepatic mRNA expression of AHR2 is high and its promoter is hypomethylated. Taken together, our results suggest that genetic resistance to contaminants in NBH fish is not due to altered methylation of AHR promoter regions, but that promoter methylation may control tissue-specific expression of AHR genes in killifish., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. In some mammalian cell lines, TCDD induces G1 cell cycle arrest, which depends on an interaction between the AHR and the retinoblastoma tumor suppressor (RB). Mammals possess one AHR, whereas fishes possess two or more AHR paralogs that differ in the domains important for AHR-RB interactions in mammals. To test the hypothesis that fish AHR paralogs differ in their ability to interact with RB, we cloned RB cDNA from Atlantic killifish, Fundulus heteroclitus, and studied the interactions of killifish RB protein with killifish AHR1 and AHR2. In coimmunoprecipitation experiments, in vitro-expressed killifish RB coprecipitated with both AHR1 and AHR2. Consistent with these results, both killifish AHR1 and AHR2 interacted with RB in mammalian two-hybrid assays. These results suggest that both fish AHR1 and AHR2 paralogs may have the potential to influence cell proliferation through interactions with RB.

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

The aryl hydrocarbon receptor (AHR) repressor (AHRR) inhibits AHR-mediated transcription and has been associated with reproductive dysfunction and tumorigenesis in humans. Previous studies have characterized the repressor function of AHRRs from mice and fish, but the human AHRR ortholog (AHRR(715)) appeared to be nonfunctional in vitro. Here, we report a novel human AHRR cDNA (AHRRDelta8) that lacks exon 8 of AHRR(715). AHRRDelta8 was the predominant AHRR form expressed in human tissues and cell lines. AHRRDelta8 effectively repressed AHR-dependent transactivation, whereas AHRR(715) was much less active. Similarly, AHRRDelta8, but not AHRR(715), formed a complex with AHR nuclear translocator (ARNT). Repression of AHR by AHRRDelta8 was not relieved by overexpression of ARNT or AHR coactivators, suggesting that competition for these cofactors is not the mechanism of repression. AHRRDelta8 interacted weakly with AHR but did not inhibit its nuclear translocation. In a survey of transcription factor specificity, AHRRDelta8 did not repress the nuclear receptor pregnane X receptor or estrogen receptor alpha but did repress hypoxia-inducible factor (HIF)-dependent signaling. AHRRDelta8-Pro(185) and -Ala(185) variants, which have been linked to human reproductive disorders, both were capable of repressing AHR or HIF. Together, these results identify AHRRDelta8 as the active form of human AHRR and reveal novel aspects of its function and specificity as a repressor.

Activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin causes altered gene expression and toxicity. The AHR repressor (AHRR) inhibits AHR signaling through a proposed mechanism involving competition with AHR for dimerization with AHR nuclear translocator (ARNT) and binding to AHR-responsive enhancer elements (AHREs). We sought to delineate the relative roles of competition for ARNT and AHREs in the mechanism of repression. In transient transfections in which AHR2-dependent transactivation was repressed by AHRR1 or AHRR2, increasing ARNT expression failed to reverse the repression, suggesting that AHRR inhibition of AHR signaling does not occur through sequestration of ARNT. An AHRR1 point mutant (AHRR1-Y9F) that could not bind to AHREs but that retained its nuclear localization was only slightly reduced in its ability to repress AHR2, demonstrating that AHRR repression does not occur solely through competition for AHREs. When both proposed mechanisms were blocked (AHRR1-Y9F plus excess ARNT), AHRR remained functional. AHRR1 neither blocked AHR nuclear translocation nor reduced the levels of AHR2 protein. Experiments using AHRR1 C-terminal deletion mutants showed that amino acids 270 to 550 are dispensable for repression. These results demonstrate that repression of AHR transactivation by AHRR involves the N-terminal portion of AHRR; does not involve competition for ARNT; and does not require binding to AHREs, although AHRE binding can contribute to the repression. We propose a mechanism of AHRR action involving "transrepression" of AHR signaling through protein-protein interactions rather than by inhibition of the formation or DNA binding of the AHR-ARNT complex.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce various toxic effects through the aryl hydrocarbon receptor (AhR) signaling pathway. Here, we investigated the structural and functional characteristics and molecular evolution of multiple AhRs in black-footed albatross (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo). We report the complementary DNA sequences of two distinct AhRs, designated AhR1 and AhR2, from these species as well as the identification of an AhR2-like gene sequence from the chicken genome database. Phylogenetic analysis reveals that avian AhR1 and AhR2 are orthologous to mammalian AhR1 and fish AhR2, respectively, supporting the hypothesis that an ancestral AhR gene underwent a tandem duplication prior to the divergence of fish and tetrapod lineages. In vitro-expressed AhR1 and AhR2 isoforms from both albatross and cormorant exhibited specific binding to [3H]TCDD, as assessed by velocity sedimentation. An in vitro reporter gene transactivation assay revealed that both AhR1 and AhR2 are transcriptionally active, but AhR2 appears to have reduced transcriptional efficacy. Hepatic messenger RNA expression level of cormorant AhR1 was greater than that of AhR2. Together, these results suggest that AhR1 is the dominant form of avian AhRs, in contrast to fish, in which AhR2 is the major form. Comparative analysis of AhR diversity and gene synteny among chicken, zebrafish, and human suggests that additional, independent AhR duplications have occurred in the fish and tetrapod lineages following the initial tandem duplication on the ancestral chromosome. The identification and characterization of avian AhR1 and AhR2 provide new insight into the evolution of AhR structure and function in vertebrates.

Ligand-activated receptors are well-known targets of environmental chemicals that disrupt endocrine signaling. Genomic approaches are providing new opportunities to understand the comparative biology and molecular evolution of these receptors. One example of this is the aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factor through which planar aromatic hydrocarbons cause altered gene expression and toxicity. In contrast to humans and other mammals, which possess a single AHR, teleosts such as the Atlantic killifish (Fundulus heteroclitus) have at least two AHRs (AHR1 and AHR2). Analysis of sequenced genomes has revealed additional, unexpected AHR diversity in non-mammalian vertebrates, including the chicken Gallus gallus (three predicted AHR genes), bony fishes such as the pufferfish Takifugu (formerly Fugu) rubripes (five AHR genes) and zebrafish Danio rerio (three AHR genes), and cartilaginous fishes such as the spiny dogfish Squalus acanthias (three AHR genes). In contrast, invertebrates appear to possess single AHRs that do not bind typical ligands of vertebrate AHRs. We suggest that AHR diversity in vertebrates arose through both gene and whole-genome duplications combined with lineage-specific gene loss, and that sensitivity to the developmental toxicity of planar aromatic hydrocarbons may have had its origin in the evolution of the ligand-binding capacity of the AHR in the chordate lineage. Comparative molecular and genomic studies are providing new insights into AHR diversity and function in non-mammalian species, revealing additional complexity in mechanisms by which environmental chemicals interfere with receptor-dependent signaling., ((c) 2006 Wiley-Liss, Inc.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons (HAHs) are highly toxic to most vertebrate animals, but there are dramatic differences in sensitivity among species and strains. Aquatic birds including the common tern (Sterna hirundo) are highly exposed to HAHs in the environment, but are up to 250-fold less sensitive to these compounds than the typical avian model, the domestic chicken (Gallus gallus). The mechanism of HAH toxicity involves altered gene expression subsequent to activation of the aryl hydrocarbon receptor (AHR), a basic helix-loop-helix-PAS transcription factor. AHR polymorphisms underlie mouse strain differences in sensitivity to HAHs and polynuclear aromatic hydrocarbons, but the role of the AHR in species differences in HAH sensitivity is not well understood. Here, we show that although chicken and tern AHRs both exhibit specific binding of [3H]TCDD, the tern AHR has a lower binding affinity and exhibits a reduced ability to support TCDD-dependent transactivation as compared to AHRs from chicken or mouse. We further show through use of chimeric AHR proteins and site-directed mutagenesis that the difference between the chicken and tern AHRs resides in the ligand-binding domain and that two amino acids (Val-325 and Ala-381) are responsible for the reduced activity of the tern AHR. Other avian species with reduced sensitivity to HAHs also possess these residues. These studies provide a molecular understanding of species differences in sensitivity to dioxin-like compounds and suggest an approach to using the AHR as a marker of dioxin susceptibility in wildlife.

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.

The aryl hydrocarbon receptor (AhR) is an environmental carcinogen-activated transcription factor associated with tumorigenesis. High levels of apparently active AhR characterize a variety of tumors, even in the absence of environmental ligands. Despite this association between transformation and AhR upregulation, little is known of the transcriptional consequences of constitutive AhR activation. Here, the effects of constitutively active and environmental ligand-induced AhR on c-myc, an oncogene whose promoter contains six AhR-binding sites (AhREs (aryl hydrocarbon response elements)), were investigated. A reporter containing the human c-myc promoter, with its six AhREs and two NF-kappaB-binding sites, was constructed. This vector, and variants with deletions in the NF-kappaB and/or AhR-binding sites, was transfected into a human breast cancer cell line, Hs578T, which expresses high levels of apparently active, nuclear AhR. Results indicate that: (1) the AhR constitutively binds the c-myc promoter; (2) there is a low but significant baseline level of c-myc promoter activity, which is not regulated by NF-kappaB and is not affected by an environmental AhR ligand; (3) deletion of any one of the AhREs has no effect on constitutive reporter activity, while deletion of all six increases reporter activity approximately fivefold; (4) a similar increase in reporter activity occurs when constitutively active AhR is suppressed by transfection with an AhR repressor plasmid (AhRR); (5) AhRR transfection significantly increases background levels of endogenous c-myc mRNA and c-Myc protein. These results suggest that the AhR influences the expression of c-Myc, a protein critical to malignant transformation.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates gene expression following activation by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or a variety of other synthetic and natural compounds. Previous studies have identified two AHR genes, AHR1 and AHR2, in zebrafish (Danio rerio), a widely used model species for studying vertebrate development and an emerging model in developmental toxicology. Zebrafish AHR2 binds TCDD with high affinity, is transcriptionally active and has a major role in mediating the developmental toxicity of TCDD. Zebrafish AHR1 lacks the ability to bind TCDD and activate transcription, and has no known function. In the present study, we report a new zebrafish AHR, designated AHR1B, which shares 34% amino acid sequence identity with AHR1 (AHR1A). The ahr1b gene resides on chromosome 22, adjacent to ahr2, whereas the ahr1a gene is located on chromosome 16. AHR1B is expressed in embryos as early as 24 hours post-fertilization and increases through the next 2 days, but expression is not inducible by TCDD. In contrast with the previously identified AHR1A, in vitro-expressed AHR1B protein exhibits specific, high-affinity binding of [3H]TCDD. Furthermore, AHR1B is able to activate the transcription of a reporter gene under the control of AHR response elements with an efficacy comparable with that of AHR2, but with a higher EC50. We speculate that AHR1B may have a physiological role, such as in embryonic development, whereas AHR2 mediates the response to xenobiotics.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The recently identified AHR repressor (AHRR) forms a negative feedback loop with the AHR. We investigated AHRR structure, function, evolution, and regulation in zebrafish, a powerful model in developmental biology and toxicology. We identified and cloned two distinct AHRR cDNAs that encode predicted proteins of 550 (AHRR1) and 573 (AHRR2) amino acids. The ahrr1 and ahrr2 genes map to zebrafish chromosomes 24 and 2, respectively, both of which share conserved synteny with human chromosome 5, the location of human AHRR. Mapping and phylogenetic analysis show that AHRR1 and AHRR2 are co-orthologs of the mammalian AHRR. In transient transfection assays, AHRR1 and AHRR2 repressed constitutive and TCDD-inducible transactivation by AHR2. Expression of both AHRR mRNAs was induced in ZF-L cells by AHR agonists but not by non-agonists. TCDD induced AHRR1 and AHRR2 expression in a dose-dependent manner in ZF-L cells, with EC50 values similar to those for induction of CYP1A. Both AHRRs were expressed and induced by TCDD in zebrafish embryos. Thus, zebrafish possess duplicate AHR-regulated AHRR paralogs that act in a negative feedback loop to repress the AHR signaling pathway.

Enzymes in the cytochrome P450 gene family 1 (CYP1) catalyze the metabolic activation of numerous hydrocarbon carcinogens and various natural compounds. CYP1 family members have been identified in several vertebrates, including fish, amphibians, birds, and mammals, and are inducible by aromatic hydrocarbons acting through the aryl hydrocarbon receptor (AHR). Together with its heterodimeric partner ARNT, the ligand-bound AHR binds conserved xenobiotic response elements (XREs) near the promoter of CYP1A and other genes. However, some populations of the Atlantic killifish Fundulus heteroclitus inhabiting highly contaminated sites are refractory to CYP1A induction by aromatic hydrocarbons. To better understand the mechanisms underlying this phenomenon, we are characterizing the AHR-CYP1A signaling pathway in this species. We report here the characterization of a genomic clone containing the 5(') end of the wild-type F. heteroclitus CYP1A gene. The 5(') coding sequence matches that of the F. heteroclitus CYP1A cDNA reported earlier [Comp. Biochem. Physiol. 121C (1998) 231]. Consistent with its inducibility by AHR agonists, the CYP1A gene contains three consensus XREs (5(')CACGC3(')) within 1.6 kb of the putative transcriptional start site. When oligonucleotides containing each of these sites were analyzed in an electrophoretic mobility shift assay, one of these showed a strong, TCDD-inducible mobility shift in the presence of in vitro expressed mouse AHR protein. These sequence data and initial functional characterization provide a valuable tool for the study of genetic variations in CYP1A expression and activity in sensitive and resistant populations. These studies may ultimately shed light on the importance of P4501A activity in xenobiotic toxicity.