The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior.

The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48 h postfertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly coexpressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1's role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1's exon, and exposed wildtype and mutants to dimethyl sulfoxide or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < .05, log2FC > 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD's overt toxicity but is likely a necessary functional member of the AHR signaling cascade.

Signaling Events Downstream of AHR Activation That Contribute to Toxic Responses: The Functional Role of an AHR-Dependent Long Noncoding RNA (slincR) Using the Zebrafish Model.

BACKGROUND: A structurally diverse group of chemicals, including dioxins [e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] and polycyclic aromatic hydrocarbons (PAHs), can xenobiotically activate the aryl hydrocarbon receptor (AHR) and contribute to adverse health effects in humans and wildlife. In the zebrafish model, repression of sox9b has a causal role in several AHR-mediated toxic responses, including craniofacial cartilage malformations; however, the mechanism of sox9b repression remains unknown. We previously identified a long noncoding RNA, sox9b long intergenic noncoding RNA (slincR), which is increased (in an AHR-dependent manner) by multiple AHR ligands and is required for the AHR-activated repression of sox9b. OBJECTIVE: Using the zebrafish model, we aimed to enhance our understanding of the signaling events downstream of AHR activation that contribute to toxic responses by identifying: a) whether slincR is enriched on the sox9b locus, b) slincR's functional contributions to TCDD-induced toxicity, c) PAHs that increase slincR expression, and d) mammalian orthologs of slincR. METHODS: We used capture hybridization analysis of RNA targets (CHART), qRT-PCR, RNA sequencing, morphometric analysis of cartilage structures, and hemorrhaging screens. RESULTS: The slincR transcript was enriched at the 5' untranslated region (UTR) of the sox9b locus. Transcriptome profiling and human ortholog analyses identified processes related to skeletal and cartilage development unique to TCDD-exposed controls, and angiogenesis and vasculature development unique to TCDD-exposed zebrafish that were injected with a splice-blocking morpholino targeting slincR. In comparison to TCDD exposed control morphants, slincR morphants exposed to TCDD resulted in abnormal cartilage structures and a smaller percentage of animals displaying the hemorrhaging phenotype. In addition, slincR expression was significantly increased in six out of the sixteen PAHs we screened. CONCLUSION: Our study establishes that in zebrafish, slincR is recruited to the sox9b 5' UTR to repress transcription, can regulate cartilage development, has a causal role in the TCDD-induced hemorrhaging phenotype, and is up-regulated by multiple environmentally relevant PAHs. These findings have important implications for understanding the ligand-specific mechanisms of AHR-mediated toxicity. https://doi.org/10.1289/EHP3281.

Elucidating Gene-by-Environment Interactions Associated with Differential Susceptibility to Chemical Exposure.

BACKGROUND: Modern societies are exposed to vast numbers of potentially hazardous chemicals. Despite demonstrated linkages between chemical exposure and severe health effects, there are limited, often conflicting, data on how adverse health effects of exposure differ across individuals. OBJECTIVES: We tested the hypothesis that population variability in response to certain chemicals could elucidate a role for gene-environment interactions (GxE) in differential susceptibility. METHODS: High-throughput screening (HTS) data on thousands of chemicals in genetically heterogeneous zebrafish were leveraged to identify a candidate chemical (Abamectin) with response patterns indicative of population susceptibility differences. We tested the prediction by generating genome-wide sequence data for 276 individual zebrafish displaying susceptible (Affected) vs. resistant (Unaffected) phenotypes following identical chemical exposure. RESULTS: We found GxE associated with differential susceptibility in the sox7 promoter region and then confirmed gene expression differences between phenotypic response classes. CONCLUSIONS: The results for Abamectin in zebrafish demonstrate that GxE associated with naturally occurring, population genetic variation play a significant role in mediating individual response to chemical exposure. https://doi.org/10.1289/EHP2662.

Investigating the application of a nitroreductase-expressing transgenic zebrafish line for high-throughput toxicity testing.

Nitroreductase enzymes are responsible for the reduction of nitro functional groups to amino functional groups, and are found in a range of animal models, zebrafish (Danio rerio) excluded. Transgenic zebrafish models have been developed for tissue-specific cell ablation, which use nitroreductase to ablate specific tissues or cell types following exposure to the non-toxic pro-drug metronidazole (MTZ). When metabolized by nitroreductase, MTZ produces a potent cytotoxin, which specifically ablates the tissue in which metabolism occurs. Uses, beyond tissue-specific cell ablation, are possible for the hepatocyte-specific Tg(l-fabp:CFP-NTR)s891 zebrafish line, including investigations of the role of nitroreductase in the toxicity of nitrated compounds. The hepatic ablation characteristics of this transgenic line were explored, in order to expand its potential uses. Embryos were exposed at 48, 72, or 96 hours post fertilization (hpf) to a range of MTZ concentrations, and the ablation profiles were compared. Ablation occurred at a 10-fold lower concentration than previously reported. Embryos were exposed to a selection of other compounds, with and without MTZ, in order to investigate alternative uses for this transgenic line. Test compounds were selected based on: their ability to undergo nitroreduction, known importance of hepatic metabolism to toxicity, and known pharmaceutical hepatotoxins. Selected compounds included nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), the PAHs retene and benzo[a]pyrene, and the pharmaceuticals acetaminophen and flutamide. The results suggest a range of potential roles of the liver in the toxicity of these compounds, and highlight the additional uses of this transgenic model in toxicity testing.

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.

Mechanistic Investigations Into the Developmental Toxicity of Nitrated and Heterocyclic PAHs.

Nitrated polycyclic aromatic hydrocarbons (NPAHs) and heterocyclic PAHs (HPAHs) are recognized environmental pollutants. However, the health risks of NPAHs and HPAHs to humans and environmental systems are not well-studied. The developmental zebrafish (Danio rerio) model was used to evaluate the toxicity of a structurally diverse set of 27 NPAHs and 10 HPAHs. The individual activity of each compound towards the aryl hydrocarbon receptor (AHR), including the role of the AHR in observed toxicity, and genetic markers of oxidative stress and cardiac toxicity were evaluated. Zebrafish embryos were exposed from 6 to 120 hours post fertilization (hpf), to a broad concentration range of individual compounds, and evaluated for 22 developmental endpoints. The potential role of AHR was determined using the transgenic Tg(cyp1a:nls-egfp) reporter zebrafish line. All compounds were screened computationally through molecular docking using a previously developed AHR models of zebrafish isoforms 1A, 1B, and 2. Some compounds did not induce observable developmental toxic responses, whereas others produced statistically significant concentration-dependent toxicity. The tested compounds also exhibited a range of predicted AHR binding and cyp1a/GFP induction patterns, including cyp1a expression in the liver, vasculature, skin, and yolk, which we determined to be due to distinct isoforms of the AHR, using morpholino oligonucleotide knockdown. Furthermore, we investigated mRNA expression of oxidative and cardiac stress genes at 48 and 120 hpf, which indicated several potential mechanisms-of-action for NPAHs. Overall, we observed a range of developmental toxicities, cyp1a/GFP expression patterns, and gene expression profiles, suggestive of several potential mechanisms of action.

Phenotype anchoring in zebrafish reveals a potential role for matrix metalloproteinases (MMPs) in tamoxifen's effects on skin epithelium.

The zebrafish is a powerful alternative model used to link phenotypes with molecular effects to discover drug mode of action. Using a zebrafish embryo-larval toxicity bioassay, we evaluated the effects of tamoxifen--a widely used anti-estrogen chemotherapeutic. Zebrafish exposed to ≥ 10 µM tamoxifen exhibited a unique necrotic caudal fin phenotype that was rapidly induced regardless of developmental life-stage when treatment was applied. To define tamoxifen's bioactivity resulting in this phenotype, targeted gene expression was used to evaluate 100 transcripts involved in tissue remodeling, calcium signaling, cell cycle and cell death, growth factors, angiogenesis and hypoxia. The most robustly misregulated transcripts in the tail were matrix metalloproteinases mmp9 and mmp13a, induced 127 and 1145 fold, respectively. Expression of c-fos, c-jun, and ap1s1 were also moderately elevated (3-7 fold), consistent with AP-1 activity--a transcription factor that regulates MMP expression. Immunohistochemistry confirmed high levels of induction for MMP13a in affected caudal fin skin epithelial tissue. The necrotic caudal fin phenotype was significantly attenuated or prevented by three functionally unique MMP inhibitors: EDTA (metal chelator), GM 6001 (broad MMP inhibitor), and SR 11302 (AP-1 transcription factor inhibitor), suggesting MMP-dependence. SR 11302 also inhibited induction of mmp9, mmp13a, and a putative MMP target, igfbp1a. Overall, our studies suggest that tamoxifen's effect is the result of perturbation of the MMP system in the skin leading to ectopic expression, cytotoxicity, and the necrotic caudal fin phenotype. These studies help advance our understanding of tamoxifen's non-classical mode of action and implicate a possible role for MMPs in tissues such as skin.

Retinoic acid-dependent regulation of miR-19 expression elicits vertebrate axis defects.

Retinoic acid (RA) is involved in multifarious and complex functions necessary for vertebrate development. RA signaling is reliant on strict enzymatic regulation of RA synthesis and metabolism. Improper spatiotemporal expression of RA during development can result in vertebrate axis defects. microRNAs (miRNAs) are also pivotal in orchestrating developmental processes. While mechanistic links between miRNAs and axial development are established, the role of miRNAs in regulating metabolic enzymes responsible for RA abundance during axis formation has yet to be elucidated. Our results uncovered a role of miR-19 family members in controlling RA metabolism through the regulation of CYP26A1 during vertebrate axis formation. Global miRNA expression profiling showed that developmental RA exposure suppressed the expression of miR-19 family members during zebrafish somitogenesis. A reporter assay confirmed that cyp26a1 is a bona fide target of miR-19 in vivo. Transient knockdown of miR-19 phenocopied axis defects caused by RA exposure. Exogenous miR-19 rescued the axis defects induced by RA exposure. Taken together, these results indicate that the teratogenic effects of RA exposure result, in part, from repression of miR-19 expression and subsequent misregulation of cyp26a1. This highlights a previously unidentified role of miR-19 in facilitating vertebrate axis development via regulation of RA signaling.

Investigating the impact of chronic atrazine exposure on sexual development in zebrafish.

Atrazine (ATZ) is a selective triazine herbicide used primarily for preemergent weed control in corn, sorghum, and sugar cane production. It is one of the most widely used herbicides in North America. Some research published over the last decade suggests that chronic exposure to environmentally relevant ATZ concentrations can adversely impact gonadal development and/or sexual differentiation in amphibians and fish, while other studies report no effect, or moderate effects. As a result, contrasting conclusions have been published regarding the potential effects of the herbicide ATZ on aquatic species. Two near-identical 4-month studies in 2009 (Study I) and 2010 (Study II) were performed investigating the potential for chronic ATZ exposure to affect zebrafish (Danio rerio) sexual development and differentiation. Zebrafish were chronically exposed to 0, 0.1, 1, 10 µM ATZ or 1 nM 17ß-estradiol (E2). Fish were histologically examined to assign gender and to evaluate potential impacts of E2 or ATZ on gonadal development. Exposure to E2 consistently resulted in a significantly higher proportion of female fish to normal male fish when compared to unexposed fish (both studies). In both studies, ATZ exposure did not significantly influence the percentage of female or male fish when compared to unexposed fish. A greater percentage of abnormally developed male fish and fish lacking differentiated gonadal tissue was observed in Study II E2 exposures but not in ATZ exposures. Together, these studies indicate that long-term exposure to ATZ at or above environmentally relevant concentrations does not significantly impact zebrafish gonadal development or sexual differentiation.

AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish.

The aryl hydrocarbon receptor (AHR) is well known for mediating the toxic effects of TCDD and has been a subject of intense research for over 30 years. Current investigations continue to uncover its endogenous and regulatory roles in a wide variety of cellular and molecular signaling processes. A zebrafish line with a mutation in ahr2 (ahr2(hu3335)), encoding the AHR paralogue responsible for mediating TCDD toxicity in zebrafish, was developed via Targeting Induced Local Lesions IN Genomes (TILLING) and predicted to express a non-functional AHR2 protein. We characterized AHR activity in the mutant line using TCDD and leflunomide as toxicological probes to investigate function, ligand binding and CYP1A induction patterns of paralogues AHR2, AHR1A and AHR1B. By evaluating TCDD-induced developmental toxicity, mRNA expression changes and CYP1A protein in the AHR2 mutant line, we determined that ahr2(hu3335) zebrafish are functionally null. In silico modeling predicted differential binding of TCDD and leflunomide to the AHR paralogues. AHR1A is considered a non-functional pseudogene as it does not bind TCCD or mediate in vivo TCDD toxicity. Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide. AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies. The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.

Sulfhydryl systems are a critical factor in the zebrafish developmental toxicity of the dithiocarbamate sodium metam (NaM).

Dithiocarbamates (DTCs) are sulfhydryls (thiol)-containing compounds, often associated with metals, and have both antioxidant and pro-oxidant abilities depending on the compound, experimental system and condition. In this study we investigated whether cell death plays a role in the manifestation of DTC-induced notochord distortions in the developing zebrafish and if thiol-containing compounds or antioxidants could modify this developmental toxicity. Sodium metam (NaM) induced notochord distortions could not be protected with the antioxidants ascorbic acid, trolox (synthetic vitamin E) or lipoic acid. However, NaM-induced distortions could be protected with co-exposure to glutathione or N-Acetyl Cysteine. Staggering the NaM and glutathione exposures in consecutive 10h developmental windows also resulted in protection. There were no discernable changes in TUNEL positive cells, a marker of apoptotic cells, at 24h post-fertilization (hpf) in NaM, dimethyl-dithiocarbamate, carbon disulfide, or neocuproine exposed embryos. Live NaM-exposed embryos incubated with acridine orange, a general stain for cell death, for 1h beginning at 11, 18 and 24hpf showed clusters of stained nuclei near the somitogenic front but not in the cells making up the notochord. Overall, induction of apoptotic pathways and widespread cell death are not involved in the manifestation of the adverse developmental outcomes following NaM exposure. However, cellular thiol status or critical sulfhydryl moieties are important considerations in the mechanisms of DTC developmental toxicity.

Ethanol-dependent toxicity in zebrafish is partially attenuated by antioxidants.

Ethanol is a well-established developmental toxicant; however, the molecular and cellular mechanism(s) of toxicity remains unclear. It has been suggested that ethanol metabolism leads to oxidative stress resulting in an increase in cell death. Alcohol developmental toxicity has not been well studied in zebrafish; however, zebrafish represent an excellent vertebrate model for investigating and understanding normal and aberrant development. To evaluate ethanol metabolism dependent toxicity, chemical inhibitors of the ethanol metabolizing enzymes were utilized. Embryos co-exposed to ethanol and a combination of ethanol metabolism inhibitors led to a significant increase in the occurrence of pericardial edema. Further, in the presence of the inhibitor mixture there was an increase in developmental malformations at lower ethanol concentrations. Cell death has been implicated as a potential explanation for ethanol-dependent toxicity. Using cell death assays, ethanol significantly increased embryonic cell death. To determine if oxidative stress underlies cardiovascular dysfunction, embryos were co-exposed to ethanol and several antioxidants. The antioxidants, glutathione and lipoic acid, partially attenuated the incidence of pericardial edema. The effectiveness of the antioxidants to protect the embryos from ethanol-induced cell death was also evaluated. The antioxidants provided no protection against cell death. Thus, ethanol-mediated pericardial edema and cell death appear to be mechanistically distinct.

Dithiocarbamates have a common toxic effect on zebrafish body axis formation.

We previously determined that the dithiocarbamate pesticide sodium metam (NaM) and its active ingredient methylisothiocyanate (MITC) were developmentally toxic causing notochord distortions in the zebrafish. In this study, developing zebrafish were exposed to isothiocyanates (ITCs), dithiocarbamates (DTCs) and several degradation products to determine the teratogenic relationship of these chemical classes at the molecular level. All dithiocarbamates tested elicited notochord distortions with notochord NOELs from <4 to 40 ppb, while none of the ITCs caused notochord distortions with the exception of MITC. Carbon disulfide (CS(2)), a common DTC degradate, also caused distortions at concentrations >200 times the DTCs. Whole mount in situ hybridization of developmental markers for collagen (collagen2a1), muscle (myoD), and body axis formation (no tail) was perturbed well after cessation of treatment with pyrolidine-DTC (PDTC), dimethyl-DTC (DMDTC), NaM, MITC, and CS(2). Therefore, distinct albeit related chemical classes share a common toxic effect on zebrafish notochord development. To test the responsiveness of the distortion to metal perturbation, five metal chelators and 2 metals were studied. The membrane permeable copper chelator neocuproine (NCu) was found to cause notochord distortions similar to DTC-related molecules. DMDTC and NCu treated animals were protected with copper, and collagen 2a1 and no tail gene expression patterns were identical to controls in these animals. PDTC, NaM, MITC, and CS(2) were not responsive to copper indicating that the chelation of metals is not the primary means by which these molecules elicit their developmental toxicity. Embryos treated with DMDTC, NaM, and NCu were rescued by adding triciaine (MS-222) which abolishes the spontaneous muscle contractions that begin at 18 hpf. In these animals, only collagen 2a1 expression showed a similar pattern to the other notochord distorting molecules. This indicates that the perturbation of no tail expression is in response to the muscle contractions distorting the notochord, while collagen 2a1 is associated with the impact of these molecules on much earlier developmental processes.