Urinary MicroRNA Biomarkers of Nephrotoxicity in Macaca fascicularis.

Drug-induced kidney injury (DIKI) refers to kidney damage resulting from the administration of medications. The aim of this project was to identify reliable urinary microRNA (miRNAs) biomarkers that can be used as potential predictors of DIKI before disease diagnosis. This study quantified a panel of six miRNAs (miRs-210-3p, 423-5p, 143-3p, 130b-3p, 486-5p, 193a-3p) across multiple time points using urinary samples from a previous investigation evaluating effects of a nephrotoxicant in cynomolgus monkeys. Exosome-associated miRNA exhibited distinctive trends when compared to miRNAs quantified in whole urine, which may reflect a different urinary excretion mechanism of miRNAs than those released passively into the urine. Although further research and mechanistic studies are required to elucidate how these miRNAs regulate signaling in disease pathways, we present, for the first time, data that several miRNAs displayed strong correlations with histopathology scores, thus indicating their potential use as biomarkers to predict the development of DIKI in preclinical studies and clinical trials. Also, these findings can potentially be translated into other non-clinical species or human for the detection of DIKI.

Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high µM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation.

Embryonic Exposure to Cigarette Smoke Extract Impedes Skeletal Development and Evokes Craniofacial Defects in Zebrafish.

Exposure to cigarette smoke represents the largest source of preventable death and disease in the United States. This may be in part due to the nature of the delayed harmful effects as well as the lack of awareness of the scope of harm presented by these products. The presence of "light" versions further clouds the harmful effects of tobacco products. While active smoking in expectant mothers may be reduced by educational and outreach campaigns, exposure to secondhand smoke is often involuntary yet may harm the developing embryo. In this study, we show that the main component of secondhand smoke, sidestream cigarette smoke, from several brands, including harm-reduction versions, triggered unsuccessful hatching at 3 dpf and reduced overall survival at 6 dpf in developing zebrafish. At non-lethal concentrations, craniofacial defects with different severity based on the cigarette smoke extract were noted by 6 dpf. All tested products, including harm-reduction products, significantly impacted cartilage formation and/or bone mineralization in zebrafish embryos, independent of whether the bones/cartilage formed from the mesoderm or neural crest. Together, these results in a model system often used to detect embryonic malformations imply that exposure of a woman to secondhand smoke while pregnant may lead to mineralization issues in the skeleton of her newborn, ultimately adding a direct in utero association to the increased fracture risk observed in children of mothers exposed to cigarette smoke.

miR133b Microinjection during Early Development Targets Transcripts of Cardiomyocyte Ion Channels and Induces Oil-like Cardiotoxicity in Zebrafish (Danio rerio) Embryos.

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 µM) or miR133b (75 µM) or (2) exposed to a treatment solution containing 5 µM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Maternal-to-zygotic transition as a potential target for niclosamide during early embryogenesis.

Niclosamide is an antihelminthic drug used worldwide for the treatment of tapeworm infections. Recent drug repurposing screens have highlighted the broad bioactivity of niclosamide across diverse mechanisms of action. As a result, niclosamide is being evaluated for a range of alternative drug-repurposing applications, including the treatment of cancer, bacterial infections, and Zika virus. As new applications of niclosamide will require non-oral delivery routes that may lead to exposure in utero, it is important to understand the mechanism of niclosamide toxicity during early stages of embryonic development. Previously, we showed that niclosamide induces a concentration-dependent delay in epiboly progression in the absence of effects on oxidative phosphorylation - a well-established target for niclosamide. Therefore, the overall objective of this study was to further examine the mechanism of niclosamide-induced epiboly delay during zebrafish embryogenesis. Based on this study, we found that (1) niclosamide exposure during early zebrafish embryogenesis resulted in a decrease in yolk sac integrity with a concomitant decrease in the presence of yolk sac actin networks and increase in cell size; (2) within whole embryos, niclosamide exposure did not alter non-polar metabolites and lipids, but significantly altered amino acids specific to aminoacyl-tRNA biosynthesis; (3) niclosamide significantly altered transcripts related to translation, transcription, and mRNA processing pathways; and (4) niclosamide did not significantly alter levels of rRNA and tRNA. Overall, our findings suggest that niclosamide may be causing a systemic delay in embryonic development by disrupting the translation of maternally-supplied mRNAs, an effect that may be mediated through disruption of aminoacyl-tRNA biosynthesis.

Diphenyl Phosphate-Induced Toxicity During Embryonic Development.

Diphenyl phosphate (DPHP) is an aryl phosphate ester (APE) used as an industrial catalyst and chemical additive and is the primary metabolite of flame retardant APEs, including triphenyl phosphate (TPHP). Minimal DPHP-specific toxicity studies have been published despite ubiquitous exposure within human populations following metabolism of TPHP and other APEs. Therefore, the objective of this study was to determine the potential for DPHP-induced toxicity during embryonic development. Using zebrafish as a model, we found that DPHP significantly increased the distance between the sinus venosus and bulbus arteriosis (SV-BA) at 72 h postfertilization (hpf) following initiation of exposure before and after cardiac looping. Interestingly, pretreatment with d-mannitol mitigated DPHP-induced effects on SV-BA length despite the absence of DPHP effects on pericardial area, suggesting that DPHP-induced cardiac defects are independent of pericardial edema formation. Using mRNA-sequencing, we found that DPHP disrupts pathways related to mitochondrial function and heme biosynthesis; indeed, DPHP significantly decreased hemoglobin levels in situ at 72 hpf following exposure from 24 to 72 hpf. Overall, our findings suggest that, similar to TPHP, DPHP impacts cardiac development, albeit the potency of DPHP is significantly less than TPHP within developing zebrafish.

mRNA-miRNA-Seq Reveals Neuro-Cardio Mechanisms of Crude Oil Toxicity in Red Drum ( Sciaenops ocellatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil can cause global gene dysregulation and developmental impairment in fish. However, the mechanisms that alter gene regulation are not well understood. In this study, larval red drum ( Sciaenops ocellatus) were exposed to water accommodated fractions of source oil (6.8, 13.7, and 35.9 µg/L total PAHs) and weathered slick oil (4.7, 8.1, and 18.0 µg/L total PAHs) from the Deepwater Horizon (DWH) oil spill. The global mRNA-microRNA functional networks associated with the toxicity of DWH oil were explored by next-generation sequencing and in-depth bioinformatics analyses. Both source and slick oil significantly altered the expression of miR-18a, miR-27b, and miR-203a across all exposure concentrations. Consistent with the observed concentration-dependent morphological changes, the target mRNAs of these microRNAs were predominantly involved in neuro-cardio system development processes and associated key signaling pathways such as axonal guidance signaling, cAMP-response-element-binding protein signaling in neurons, calcium signaling, and nuclear-factor-of-activated T cells signaling in cardiac hypertrophy. The results indicated that the developmental toxicity of crude oil may result from the abnormal expression of microRNAs and associated target genes, especially for the nervous system. Moreover, we provide a case study for systematic toxicity evaluation leveraging mRNA-microRNA-seq data using nonmodel species.

Complex Interplay Among Nuclear Receptor Ligands, Cytosine Methylation, and the Metabolome in Driving Tris(1,3-dichloro-2-propyl)phosphate-Induced Epiboly Defects in Zebrafish.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production-volume organophosphate flame retardant (OPFR) that induces epiboly defects during zebrafish embryogenesis, leading to the disruption of dorsoventral patterning. Therefore, the objectives of this study were to (1) identify the potential mechanisms involved in TDCIPP-induced epiboly defects and (2) determine whether coexposure to triphenyl phosphate (TPHP)-an OPFR commonly detected with TDCIPP-enhances or mitigates epiboly defects. Although TDCIPP-induced epiboly defects were not associated with adverse impacts on cytoskeletal protein abundance in situ, the coexposure of embryos to TPHP partially blocked TDCIPP-induced epiboly defects. As nuclear receptors are targets for both TPHP and TDCIPP, we exposed the embryos to TDCIPP in the presence or absence of 69 nuclear receptor ligands and, similar to TPHP, found that ciglitazone (a peroxisome proliferator-activated receptor γ agonist) and 17ß-estradiol (E2; an estrogen receptor α agonist) nearly abolished TDCIPP-induced epiboly defects. Moreover, E2 and ciglitazone mitigated TDCIPP-induced effects on CpG hypomethylation within the target loci prior to epiboly, and ciglitazone altered TDCIPP-induced effects on the abundance of two polar metabolites (acetylcarnitine and cytidine-5-diphosphocholine) during epiboly. Overall, our results point to a complex interplay among nuclear receptor ligands, cytosine methylation, and the metabolome in both the induction and mitigation of epiboly defects induced by TDCIPP.

Tris(1,3-dichloro-2-propyl) Phosphate Exposure During the Early-Blastula Stage Alters the Normal Trajectory of Zebrafish Embryogenesis.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate flame retardant used around the world. Within zebrafish, we previously showed that initiation of TDCIPP exposure during cleavage (0.75 h post-fertilization, hpf) results in epiboly disruption at 6 hpf, leading to dorsalized embryos by 24 hpf, a phenotype that mimics the effects of dorsomorphin (DMP), a bone morphogenetic protein (BMP) antagonist that dorsalizes embryos in the absence of epiboly defects. The objective of this study was to (1) investigate the role of BMP signaling in TDCIPP-induced toxicity during early embryogenesis, (2) identify other pathways and processes targeted by TDCIPP, and (3) characterize the downstream impacts of early developmental defects. Using zebrafish as a model, we first identified a sensitive window for TDCIPP-induced effects following exposure initiation at 0.75 hpf. We then investigated the effects of TDCIPP on the transcriptome during the first 24 h of development using mRNA sequencing and amplicon sequencing. Finally, we relied on whole-mount immunohistochemistry, dye-based labeling, and morphological assessments to study abnormalities later in embryonic development. Overall, our data suggest that the initiation of TDCIPP exposure during early blastula alters the normal trajectory of early embryogenesis by inducing gastrulation defects and aberrant germ-layer formation, leading to abnormal tissue and organ development within the embryo.

Structural disruption increases toxicity of graphene nanoribbons.

The increased utilization of graphene nanoribbons (GNRs) for biomedical and material science applications necessitates the thorough evaluation of potential toxicity of these materials under both intentional and accidental exposure scenarios. We here investigated the effects of structural disruption of GNRs (induced by low-energy bath and high-energy probe sonication) to in vitro (human cell lines), and in vivo (Oryzias latipes embryo) biological systems. Our results demonstrate that low concentration (20 µg ml(-1) ) suspensions of GNRs prepared by as little as 1 min of probe sonication can cause significant decreases in the overall metabolic state of cells in vitro, and increased embryo/larval mortality in vivo, as compared to bath sonicated or unsonicated suspensions. Structural analysis indicates that probe sonication leads to disruption in GNR structure and production of smaller carbonaceous debris, which may be the cause of the toxicity observed. These results point out the importance of assessing post-production structural modifications for any application using nanomaterials.

Single cell sequencing of zebrafish kidney marrows reveals AHR2-dependent endogenous regulation of hematopoiesis.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a wide range of biological and toxicological responses. While largely studied in ligand-activated toxicant responses, AHR also plays important roles in endogenous physiological processes. We leveraged single cell sequencing and an AHR2 knockout zebrafish line to investigate the role of AHR2 in regulating hematopoiesis (production and differentiation of red and white blood cells from hematopoietic stem cells). Our objectives were to determine if absence of AHR2- 1) alters proportions of immune cell populations and/or 2) impacts gene expression within individual immune cell types. We dissected kidney marrow (organ of hematopoiesis in zebrafish) from adult wildtype and AHR2 knockout zebrafish (N=3/genotype), isolated single cells and sequenced ~ 5000 cells/sample (10X Genomics). We identified 14 cell clusters representing the expected major blood (erythrocytes, thrombocytes), immune (B cells, macrophages, lymphoid cells, granulocytes, etc), progenitors and kidney cell populations. We focused our analyses only on the progenitor and mature immune cell types. While there were no genotype-specific differences in proportion of individual cell types, gene expression differences were observed within several cell types. For known genes, such as rrm2, changes were up to 2000-fold, signifying their importance in AHR2-hematopoesis interaction. Several of the known genes are also identified as markers of carcinoma cells for an array of cancer types. However, many of the dysregulated genes are poorly annotated, limiting our ability to examine biological processes and pathways dysregulated on AHR2 mutation. Nevertheless, our study indicates that AHR2 plays an important endogenous role in hematopoiesis. Future work will focus on better characterizing anatomy of dysregulated genes and their functions in hematopoiesis.

Flame retardant tetrabromobisphenol A (TBBPA) disrupts histone acetylation during zebrafish maternal-to-zygotic transition.

3,3',5.5'-Tetrabromobisphenol A (TBBPA) is a widely used brominated flame-retardant utilized in the production of electronic devices and plastic paints. The objective of this study is to use zebrafish as a model and determine the effects of TBBPA exposure on early embryogenesis. We initiated TBBPA exposures (0, 10, 20 and 40µM) at 0.75 h post fertilization (hpf) and monitored early developmental events such as cleavage, blastula and epiboly that encompass maternal-to-zygotic transition (MZT) and zygotic genome activation (ZGA). Our data revealed that TBBPA exposures induced onset of developmental delays by 3 hpf (blastula). By 5.5 hpf (epiboly), TBBPA-exposed (10-20 µM) embryos showed concentration-dependent developmental lag by up to 3 stages or 100% mortality at 40 µM. Embryos exposed to sublethal TBBPA concentrations from 0.75-6 hpf and raised in clean water to 120 hpf showed altered larval photomotor response (LPR), suggesting a compromised developmental health. To examine the genetic basis of TBBPA-induced delays, we conducted mRNA-sequencing on embryos exposed to 0 or 40 µM TBBPA from 0.75 hpf to 2, 3.5 or 4.5 hpf. Read count data showed that while TBBPA exposures had no overall impacts on maternal or maternal-zygotic genes, collective read counts for zygotically activated genes were lower in TBBPA treatment at 4.5 hpf compared to time-matched controls, suggesting that TBBPA delays ZGA. Gene ontology assessments for both time- and stage-matched differentially expressed genes revealed TBBPA-induced inhibition of chromatin assembly- a process regulated by histone modifications. Since acetylation is the primary histone modification system operant during early ZGA, we immunostained embryos with an H3K27Ac antibody and demonstrated reduced acetylation in TBBPA-exposed embryos. Leveraging in silico molecular docking studies and in vitro assays, we also showed that TBBPA potentially binds to P300- a protein that catalyzes acetylation- and inhibits P300 activity. Finally, we co-exposed embryos to 20 µM TBBPA and 50 µM n-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) -a histone acetyltransferase activator that promotes histone acetylation- and showed that TBBPA-CTPB co or pre-exposures significantly reversed TBBPA-only developmental delays, suggesting that TBBPA-induced phenotypes are indeed driven by repression of histone acetylation. Collectively, our work demonstrates that TBBPA disrupts ZGA and early developmental morphology, potentially by inhibiting histone acetylation. Future studies will focus on mechanisms of TBBPA-induced chromatin modifications.

A CRISPR-Cas9 mutation in sox9b long intergenic noncoding RNA (slincR) affects zebrafish development, behavior, and regeneration.

The role of long noncoding RNAs (lncRNAs) regulators of toxicological responses to environmental chemicals is gaining prominence. Previously, our laboratory discovered an lncRNA, sox9b long intergenic noncoding RNA (slincR), that is activated by multiple ligands of aryl hydrocarbon receptor (AHR). Within this study, we designed a CRISPR-Cas9-mediated slincR zebrafish mutant line to better understand its biological function in presence or absence of a model AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The slincRosu3 line contains an 18 bp insertion within the slincR sequence that changes its predicted mRNA secondary structure. Toxicological profiling showed that slincRosu3 is equally or more sensitive to TCDD for morphological and behavioral phenotypes. Embryonic mRNA-sequencing showed differential responses of 499 or 908 genes in slincRosu3 in absence or presence of TCDD Specifically, unexposed slincRosu3 embryos showed disruptions in metabolic pathways, suggesting an endogenous role for slincR. slincRosu3 embryos also had repressed mRNA levels of sox9b-a transcription factor that slincR is known to negatively regulate. Hence, we studied cartilage development and regenerative capacity-both processes partially regulated by sox9b. Cartilage development was disrupted in slincRosu3 embryos both in presence and absence of TCDD. slincRosu3 embryos also displayed a lack of regenerative capacity of amputated tail fins, accompanied by a lack of cell proliferation. In summary, using a novel slincR mutant line, we show that a mutation in slincR can have widespread impacts on gene expression and structural development endogenously and limited, but significant impacts in presence of AHR induction that further highlights its importance in the developmental process.

Zebrafish Behavioral Assays in Toxicology.

Zebrafish behavioral assays are commonly used to identify and study environmental stressors that elicit adverse effects on neurobehavior. Behavioral assay platforms are available for multiple life stages (embryonic, juvenile, and adults) and are robust in detecting stressor-induced acute effects on neurodevelopment as well as long term deficits in sensory mechanisms, social behavior, learning, memory, and neurodegenerative diseases. Within this chapter, we present an overview of zebrafish behavioral assays that are commonly used to study environmental neurotoxicants.

Transcriptomic and Long-Term Behavioral Deficits Associated with Developmental 3.5 GHz Radiofrequency Radiation Exposures in Zebrafish.

The rapid deployment of the fifth-generation (5G) spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, concerns among the public about the safety and health effects of radiofrequency radiations (RFRs) emitted from the newer-generation cell phone frequencies remain, partly due to the lack of robust scientific data. Previously, we used developmental zebrafish to model the bioactivity of 3.5 GHz RFR, a frequency used by 5G-enabled cell phones, in a novel RFR exposure chamber. With RFR exposures from 6 h post-fertilization (hpf) to 48 hpf, we observed that, despite no teratogenic effects, embryos showed subtle hypoactivity in a startle response behavior assay, suggesting abnormal sensorimotor behavior. This study builds upon the previous one by investigating the transcriptomic basis of RFR-associated behavior effects and their persistence into adulthood. Using mRNA sequencing, we found a modest transcriptomic disruption at 48 hpf, with 28 differentially expressed genes. KEGG pathway analysis showed that biochemical pathways related to metabolism were significantly perturbed. Embryos were grown to adulthood, and then a battery of behavioral assays suggested subtle but significant abnormal responses in RFR-exposed fish across the different assays evaluated that suggest potential long-term behavioral effects. Overall, our study suggests the impacts of RFRs on the developing brain, behavior, and the metabolome should be further explored.

Utilizing Zebrafish Embryos to Reveal Disruptions in Dorsoventral Patterning.

Dorsoventral (DV) patterning is a key landmark of embryonic development that is primarily regulated by bone morphogenetic protein (BMP) signaling. Disruption of DV patterning can result in downstream effects on cell specification and organogenesis. Zebrafish embryos have been extensively used to understand signaling pathways that regulate DV patterning because zebrafish embryos develop ex utero and, in contrast to mammalian embryos, which develop in utero, can be observed in real time using brightfield and fluorescence microscopy. Embryos with disrupted DV patterning are either dorsalized or ventralized, with lack of development of head or trunk/tail structures, respectively. Although these phenotypes are typically accompanied by effects on BMP signaling, exceptions exist where some drugs or environmental chemicals can disrupt DV patterning in the absence of effects on BMP signaling. Therefore, assessments of DV patterning should be accompanied by BMP signaling-specific readouts to confirm the role of BMP disruption. Here, we describe an exposure paradigm and steps for phenotyping zebrafish embryos for two types of DV defects, dorsalization and ventralization, with a range of severities. In addition, we describe a strategy for whole-mount immunohistochemistry of zebrafish embryos with an antibody specific for phospho-SMAD 1/5/9 (pSMAD 1/5/9), as disruption in pSMAD 1/5/9 localization is indicative of an effect on BMP signaling. Taken together, these protocols describe an initial strategy for evaluating DV patterning defects under various experimental conditions and confirming BMP-mediated DV patterning disruptions, which can be followed by additional studies that aim to uncover mechanisms leading to these adverse phenotypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Phenotyping for dorsalization and ventralization Basic Protocol 2: Whole-mount immunohistochemistry with antibody to phospho-SMAD 1/5/9.

Phenotypically Anchored mRNA and miRNA Expression Profiling in Zebrafish Reveals Flame Retardant Chemical Toxicity Networks.

The ubiquitous use of flame retardant chemicals (FRCs) in the manufacture of many consumer products leads to inevitable environmental releases and human exposures. Studying toxic effects of FRCs as a group is challenging since they widely differ in physicochemical properties. We previously used zebrafish as a model to screen 61 representative FRCs and showed that many induced behavioral and teratogenic effects, with aryl phosphates identified as the most active. In this study, we selected 10 FRCs belonging to diverse physicochemical classes and zebrafish toxicity profiles to identify the gene expression responses following exposures. For each FRC, we executed paired mRNA-micro-RNA (miR) sequencing, which enabled us to study mRNA expression patterns and investigate the role of miRs as posttranscriptional regulators of gene expression. We found widespread disruption of mRNA and miR expression across several FRCs. Neurodevelopment was a key disrupted biological process across multiple FRCs and was corroborated by behavioral deficits. Several mRNAs (e.g., osbpl2a) and miRs (e.g., mir-125b-5p), showed differential expression common to multiple FRCs (10 and 7 respectively). These common miRs were also predicted to regulate a network of differentially expressed genes with diverse functions, including apoptosis, neurodevelopment, lipid regulation and inflammation. Commonly disrupted transcription factors (TFs) such as retinoic acid receptor, retinoid X receptor, and vitamin D regulator were predicted to regulate a wide network of differentially expressed mRNAs across a majority of the FRCs. Many of the differential mRNA-TF and mRNA-miR pairs were predicted to play important roles in development as well as cancer signaling. Specific comparisons between TBBPA and its derivative TBBPA-DBPE showed contrasting gene expression patterns that corroborated with their phenotypic profiles. The newer generation FRCs such as IPP and TCEP produced distinct gene expression changes compared to the legacy FRC BDE-47. Our study is the first to establish a mRNA-miR-TF regulatory network across a large group of structurally diverse FRCs and diverse phenotypic responses. The purpose was to discover common and unique biological targets that will help us understand mechanisms of action for these important chemicals and establish this approach as an important tool for better understanding toxic effects of environmental contaminants.

Effects of Phenanthrene Exposure on Cholesterol Homeostasis and Cardiotoxicity in Zebrafish Embryos.

Polycyclic aromatic hydrocarbons (PAHs) are pervasive pollutants in aquatic ecosystems, and developing fish embryos are especially sensitive to PAH exposure. Exposure to crude oil or phenanthrene (a reference PAH found in oil) produces an array of gross morphological abnormalities in developing fish embryos, including cardiotoxicity. Recently, studies utilizing transcriptomic analyses in several oil-exposed fish embryos found significant changes in the abundance of transcripts involved in cholesterol biosynthesis. Given the vital role of cholesterol availability in embryonic heart development, we hypothesized that cholesterol dysregulation in early development contributes to phenanthrene-induced cardiotoxicity. We exposed zebrafish embryos to 12 or 15 µM phenanthrene from 6 to 72 h post fertilization (hpf) and demonstrated that, in conjunction with pericardial edema and bradycardia, several genes (fdft1 and hmgcra) in the cholesterol biosynthetic pathway were significantly altered. When embryos were pretreated with a cholesterol solution from 6 to 24 hpf followed by exposure to phenanthrene from 24 to 48 hpf, the effects of phenanthrene on heart rate were partially mitigated. Despite changes in gene expression, whole-mount in situ staining of cholesterol was not significantly affected in embryos exposed to phenanthrene ranging in stage from 24 to 72 hpf. However, the 2-dimensional yolk area was significantly increased with phenanthrene exposure at 72 hpf, suggesting that lipid transport from the yolk to the developing embryo was impaired. Environ Toxicol Chem 2021;40:1586-1595. © 2021 SETAC.

A Review of the Functional Roles of the Zebrafish Aryl Hydrocarbon Receptors.

Over the last 2 decades, the zebrafish (Danio rerio) has emerged as a stellar model for unraveling molecular signaling events mediated by the aryl hydrocarbon receptor (AHR), an important ligand-activated receptor found in all eumetazoan animals. Zebrafish have 3 AHRs-AHR1a, AHR1b, and AHR2, and studies have demonstrated the diversity of both the endogenous and toxicological functions of the zebrafish AHRs. In this contemporary review, we first highlight the evolution of the zebrafish ahr genes, and the characteristics of the receptors including developmental and adult expression, their endogenous and inducible roles, and the predicted ligands from homology modeling studies. We then review the toxicity of a broad spectrum of AHR ligands across multiple life stages (early stage, and adult), discuss their transcriptomic and epigenetic mechanisms of action, and report on any known interactions between the AHRs and other signaling pathways. Through this article, we summarize the promising research that furthers our understanding of the complex AHR pathway through the extensive use of zebrafish as a model, coupled with a large array of molecular techniques. As much of the research has focused on the functions of AHR2 during development and the mechanism of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) toxicity, we illustrate the need to address the considerable knowledge gap in our understanding of both the mechanistic roles of AHR1a and AHR1b, and the diverse modes of toxicity of the various AHR ligands.

High-content screening in zebrafish identifies perfluorooctanesulfonamide as a potent developmental toxicant.

Per- and polyfluoroalkyl substances (PFASs) have been used for decades within industrial processes and consumer products, resulting in frequent detection within the environment. Using zebrafish embryos, we screened 38 PFASs for developmental toxicity and revealed that perfluorooctanesulfonamide (PFOSA) was the most potent developmental toxicant, resulting in elevated mortality and developmental abnormalities following exposure from 6 to 24 h post fertilization (hpf) and 6 to 72 hpf. PFOSA resulted in a concentration-dependent increase in mortality and abnormalities, with surviving embryos exhibiting a >12-h delay in development at 24 hpf. Exposures initiated at 0.75 hpf also resulted in a concentration-dependent delay in epiboly, although these effects were not driven by a specific sensitive window of development. We relied on mRNA-sequencing to identify the potential association of PFOSA-induced developmental delays with impacts on the embryonic transcriptome. Relative to stage-matched vehicle controls, these data revealed that pathways related to hepatotoxicity and lipid transport were disrupted in embryos exposed to PFOSA from 0.75 to 14 hpf and 0.75 to 24 hpf. Therefore, we measured liver area as well as neutral lipids in 128-hpf embryos exposed to vehicle (0.1% DMSO) or PFOSA from 0.75 to 24 hpf and clean water from 24 to 128 hpf, and showed that PFOSA exposure from 0.75 to 24 hpf resulted in a decrease in liver area and increase in yolk sac neutral lipids at 128 hpf. Overall, our findings show that early exposure to PFOSA adversely impacts embryogenesis, an effect that may lead to altered lipid transport and liver development.