Formulation Controls the Potential Neuromuscular Toxicity of Polyethylene Photoproducts in Developing Zebrafish.

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.

Developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters DNA methyltransferase (dnmt) expression in zebrafish (Danio rerio).

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.

Implications of exposure route for the bioaccumulation potential of nanopolystyrene particles.

Microplastics and nanoplastics are found in marine biota across a wide range of trophic levels and environments. While a large portion of the information about plastic exposure comes from gastrointestinal (GI) data, the relevance of particle accumulation from an oral exposure compared with other types of exposure (e.g. dermal, respiratory) is unknown. To address this gap in knowledge, larval zebrafish (7 days post fertilization) were exposed to two different sizes of nanoplastics through either oral gavage or a waterborne exposure. Larvae were tracked for 48 h post exposure (hpe) to assess the migration and elimination of plastics. Larvae eliminated orally gavaged nanoplastics within 48 hpe. Oral gavage showed limited particle movement from the GI tract into other tissues. In contrast, waterborne nanoplastic-exposed larvae displayed notable fluorescence in tissues outside of the GI tract. The 50 nm waterborne-exposed larvae retained the particles past 48 hpe, and showed accumulation with neuromasts. For both sizes of plastic particles, the nanoplastics were eliminated from non-GI tract tissues by 24 hpe. Our results suggest that waterborne exposure leads to greater accumulation of plastic in comparison to oral exposure, suggesting that plastic accumulation in certain tissues is greater via routes of exposure other than oral consumption.

Developmental exposure to domoic acid targets reticulospinal neurons and leads to aberrant myelination in the spinal cord.

Harmful algal blooms (HABs) produce neurotoxins that affect human health. Developmental exposure of zebrafish embryos to the HAB toxin domoic acid (DomA) causes myelin defects, loss of reticulospinal neurons, and behavioral deficits. However, it is unclear whether DomA primarily targets myelin sheaths, leading to the loss of reticulospinal neurons, or reticulospinal neurons, causing myelin defects. Here, we show that while exposure to DomA at 2 dpf did not reduce the number of oligodendrocyte precursors prior to myelination, it led to fewer myelinating oligodendrocytes that produced shorter myelin sheaths and aberrantly wrapped neuron cell bodies. DomA-exposed larvae lacked Mauthner neurons prior to the onset of myelination, suggesting that axonal loss is not secondary to myelin defects. The loss of the axonal targets may have led oligodendrocytes to inappropriately myelinate neuronal cell bodies. Consistent with this, GANT61, a GLI1/2 inhibitor that reduces oligodendrocyte number, caused a reduction in aberrantly myelinated neuron cell bodies in DomA-exposed fish. Together, these results suggest that DomA initially alters reticulospinal neurons and the loss of axons causes aberrant myelination of nearby cell bodies. The identification of initial targets and perturbed cellular processes provides a mechanistic understanding of how DomA alters neurodevelopment, leading to structural and behavioral phenotypes.

Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos.

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ~22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNA expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5nM) for 1h at 30hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60hpf. TCDD caused strong induction of CYP1A at 36hpf (62-fold) and 60hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular development.

Exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126) Causes Widespread DNA Hypomethylation in Adult Zebrafish Testis.

Exposure to environmental toxicants during preconception has been shown to affect offspring health and epigenetic mechanisms such as DNA methylation are hypothesized to be involved in adverse outcomes. However, studies addressing the effects of exposure to environmental toxicants during preconception on epigenetic changes in gametes are limited. The objective of this study is to determine the effect of preconceptional exposure to a dioxin-like polychlorinated biphenyl (3,3',4,4',5-pentachlorobiphenyl [PCB126]) on DNA methylation and gene expression in testis. Adult zebrafish were exposed to 3 and 10 nM PCB126 for 24 h and testis tissue was sampled at 7 days postexposure for histology, DNA methylation, and gene expression profiling. Reduced representation bisulfite sequencing revealed 37 and 92 differentially methylated regions (DMRs) in response to 3 and 10 nM PCB126 exposures, respectively. Among them, 19 DMRs were found to be common between both PCB126 treatment groups. Gene ontology (GO) analysis of DMRs revealed that enrichment of terms such as RNA processing, iron-sulfur cluster assembly, and gluconeogenesis. Gene expression profiling showed differential expression of 40 and 1621 genes in response to 3 and 10 nM PCB126 exposures, respectively. GO analysis of differentially expressed genes revealed enrichment of terms related to xenobiotic metabolism, oxidative stress, and immune function. There is no overlap in the GO terms or individual genes between DNA methylation and RNA sequencing results, but functionally many of the altered pathways have been shown to cause spermatogenic defects.

Symptomatic and asymptomatic domoic acid exposure in zebrafish (Danio rerio) revealed distinct non-overlapping gene expression patterns in the brain.

Domoic acid (DA) is a naturally produced neurotoxin synthesized by marine diatoms in the genus Pseudo-nitzschia. DA accumulates in filter-feeders such as shellfish, and can cause severe neurotoxicity when contaminated seafood is ingested, resulting in Amnesic Shellfish Poisoning (ASP) in humans. Overt clinical signs of neurotoxicity include seizures and disorientation. ASP is a significant public health concern, and though seafood regulations have effectively minimized the human risk of severe acute DA poisoning, the effects of exposure at asymptomatic levels are poorly understood. The objective of this study was to determine the effects of exposure to symptomatic and asymptomatic doses of DA on gene expression patterns in the zebrafish brain. We exposed adult zebrafish to either a symptomatic (1.1 ± 0.2 µg DA/g fish) or an asymptomatic (0.31 ± 0.03 µg DA/g fish) dose of DA by intracelomic injection and sampled at 24, 48 and 168 h post-injection. Transcriptional profiling was done using Agilent and Affymetrix microarrays. Our analysis revealed distinct, non-overlapping changes in gene expression between the two doses. We found that the majority of transcriptional changes were observed at 24 h post-injection with both doses. Interestingly, asymptomatic exposure produced more persistent transcriptional effects - in response to symptomatic dose exposure, we observed only one differentially expressed gene one week after exposure, compared to 26 in the asymptomatic dose at the same time (FDR <0.05). GO term analysis revealed that symptomatic DA exposure affected genes associated with peptidyl proline modification and retinoic acid metabolism. Asymptomatic exposure caused differential expression of genes that were associated with GO terms including circadian rhythms and visual system, and also the neuroactive ligand-receptor signaling KEGG pathway. Overall, these results suggest that transcriptional responses are specific to the DA dose and that asymptomatic exposure can cause long-term changes. Further studies are needed to characterize the potential downstream neurobehavioral impacts of DA exposure.

PCB126 Exposure Revealed Alterations in m6A RNA Modifications in Transcripts Associated With AHR Activation.

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.

Hepatic Gene Expression Profiling of Atlantic Cod (Gadus morhua) Liver after Exposure to Organophosphate Flame Retardants Revealed Altered Cholesterol Biosynthesis and Lipid Metabolism.

Since the phasing out and eventual ban on the production of organohalogen flame retardants, the use of organophosphate flame retardants (OPFRs) has increased rapidly. This has led to the detection of OPFRs in various environments including the Arctic. Two of the most prevalent OPFRs found in the Arctic are tris(2-chloroisopropyl) phosphate (TCPP), and 2-ethylhexyl diphenyl phosphate (EHDPP). The impacts of exposure to OPFRs on Arctic organisms is poorly understood. The objective of the present study was to determine the effects of exposure to TCPP, EHDPP, and a mixture of OPFRs on gene expression patterns in Atlantic cod, Gadus morhua. Precision-cut liver slices from Atlantic cod in vitro were exposed to either TCPP or EHDPP alone or in a mixture and sampled at 2 different time points to quantify gene expression patterns using RNA sequencing. We exposed the liver slices to 2 concentrations of TCPP and EHDPP, one of which was chosen based on the levels found in the Arctic environment. The RNA sequencing results demonstrated differential expression of hundreds of genes in response to exposure. The genes representing cholesterol biosynthesis and lipid metabolism pathway were significantly enriched in all the treatment groups. Almost all the cholesterol biosynthesis genes were significantly down-regulated in response to OPFR exposure. The effects on these pathways could involve various physiological processes including reproduction, growth, and behavior as well as adaptation to changing temperatures. Membrane fluidity is an important adaptive mechanism among aquatic organisms. Altered cholesterol homeostasis could have long-term consequences by altering the adaptive potential of aquatic organisms to changing water temperatures, particularly those living in polar environments. These results suggest that OPFRs could have unique effects on the organisms living in the Arctic compared with other environments. Further studies are needed to understand the long-term impacts of exposure to environmentally realistic concentrations using laboratory and field-based studies. Environ Toxicol Chem 2021;40:1639-1648. © 2021 SETAC.

Developmental Exposure to Domoic Acid Disrupts Startle Response Behavior and Circuitry in Zebrafish.

Harmful algal blooms produce potent neurotoxins that accumulate in seafood and are hazardous to human health. Developmental exposure to the harmful algal bloom toxin, domoic acid (DomA), has behavioral consequences well into adulthood, but the cellular and molecular mechanisms of DomA developmental neurotoxicity are largely unknown. To assess these, we exposed zebrafish embryos to DomA during the previously identified window of susceptibility and used the well-known startle response circuit as a tool to identify specific neuronal components that are targeted by exposure to DomA. Exposure to DomA reduced startle responsiveness to both auditory/vibrational and electrical stimuli, and even at the highest stimulus intensities tested, led to a dramatic reduction of one type of startle (short-latency c-starts). Furthermore, DomA-exposed larvae had altered kinematics for both types of startle responses tested, exhibiting shallower bend angles and slower maximal angular velocities. Using vital dye staining, immunolabeling, and live imaging of transgenic lines, we determined that although the sensory inputs were intact, the reticulospinal neurons required for short-latency c-starts were absent in most DomA-exposed larvae. Furthermore, axon tracing revealed that DomA-treated larvae also showed significantly reduced primary motor neuron axon collaterals. Overall, these results show that developmental exposure to DomA targets large reticulospinal neurons and motor neuron axon collaterals, resulting in measurable deficits in startle behavior. They further provide a framework for using the startle response circuit to identify specific neural populations disrupted by toxins or toxicants and to link these disruptions to functional consequences for neural circuit function and behavior.

Gene expression and epigenetic responses of the marine Cladoceran, Evadne nordmanni, and the copepod, Acartia clausi, to elevated CO2.

Characterizing the capacity of marine organisms to adapt to climate change related drivers (e.g., pCO2 and temperature), and the possible rate of this adaptation, is required to assess their resilience (or lack thereof) to these drivers. Several studies have hypothesized that epigenetic markers such as DNA methylation, histone modifications and noncoding RNAs, act as drivers of adaptation in marine organisms, especially corals. However, this hypothesis has not been tested in zooplankton, a keystone organism in marine food webs. The objective of this study is to test the hypothesis that acute ocean acidification (OA) exposure alters DNA methylation in two zooplanktonic species-copepods (Acartia clausii) and cladocerans (Evadne nordmanii). We exposed these two species to near-future OA conditions (400 and 900 ppm pCO2) for 24 h and assessed transcriptional and DNA methylation patterns using RNA sequencing and Reduced Representation Bisulfite Sequencing (RRBS). OA exposure caused differential expression of genes associated with energy metabolism, cytoskeletal and extracellular matrix functions, hypoxia and one-carbon metabolism. Similarly, OA exposure also caused altered DNA methylation patterns in both species but the effect of these changes on gene expression and physiological effects remains to be determined. The results from this study form the basis for studies investigating the potential role of epigenetic mechanisms in OA induced phenotypic plasticity and/or adaptive responses in zooplanktonic organisms.

Transcriptomic Changes and the Roles of Cannabinoid Receptors and PPARγ in Developmental Toxicities Following Exposure to Δ9-Tetrahydrocannabinol and Cannabidiol.

Human consumption of cannabinoid-containing products during early life or pregnancy is rising. However, information about the molecular mechanisms involved in early life stage Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) toxicities is critically lacking. Here, larval zebrafish (Danio rerio) were used to measure THC- and CBD-mediated changes on transcriptome and the roles of cannabinoid receptors (Cnr) 1 and 2 and peroxisome proliferator activator receptor γ (PPARγ) in developmental toxicities. Transcriptomic profiling of 96-h postfertilization (hpf) cnr+/+ embryos exposed (6 - 96 hpf) to 4 µM THC or 0.5 µM CBD showed differential expression of 904 and 1095 genes for THC and CBD, respectively, with 360 in common. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in the THC and CBD datasets included those related to drug, retinol, and steroid metabolism and PPAR signaling. The THC exposure caused increased mortality and deformities (pericardial and yolk sac edemas, reduction in length) in cnr1-/- and cnr2-/- fish compared with cnr+/+ suggesting Cnr receptors are involved in protective pathways. Conversely, the cnr1-/- larvae were more resistant to CBD-induced malformations, mortality, and behavioral alteration implicating Cnr1 in CBD-mediated toxicity. Behavior (decreased distance travelled) was the most sensitive endpoint to THC and CBD exposure. Coexposure to the PPARγ inhibitor GW9662 and CBD in cnr+/+ and cnr2-/- strains caused more adverse outcomes compared with CBD alone, but not in the cnr1-/- fish, suggesting that PPARγ plays a role in CBD metabolism downstream of Cnr1. Collectively, PPARγ, Cnr1, and Cnr2 play important roles in the developmental toxicity of cannabinoids with Cnr1 being the most critical.

Hyperosmotic shock adaptation by cortisol involves upregulation of branchial osmotic stress transcription factor 1 gene expression in Mozambique Tilapia.

The Mozambique tilapia (Oreochromis mossambicus) is a euryhaline species that does not survive direct seawater exposure. Cortisol is involved in re-establishing electrolyte homeostasis in seawater and is thought to play a role in allowing tilapia to cope with abrupt seawater exposure, but the mechanism(s) are far from clear. Recently, osmotic stress transcription factor 1 (OSTF1) was identified as a key signaling molecule involved in hyperosmotic stress adaptation in tilapia. Consequently, we tested the hypothesis that upregulation of OSTF1 expression by cortisol is a key response for hyperosmotic stress adaptation in tilapia. Fish were exposed to different salinities over a 24h period, while a major electrolyte disturbance and mortality was observed only with full-strength seawater exposure. Therefore, we administered cocoa butter implants of cortisol (50mg/kg) intraperitoneally to tilapia maintained in fresh water and after three days exposed these fish to full-strength seawater. There was 50% mortality in the control fish upon seawater exposure, but this was abolished by cortisol treatment. Abrupt seawater exposure did not affect plasma cortisol levels, while, as expected, exogenous administration of this steroid elevated plasma cortisol levels both in fresh water and seawater. Cortisol treatment significantly induced OSTF1 gene expression in fresh water tilapia, and also enhanced further the seawater-induced OSTF1 mRNA abundance. Plasma osmolality decreased, while gill Na(+)/K(+)-ATPase activity was suppressed in the cortisol group in seawater compared to the sham group. This corresponded with a significant reduction in gill ionocyte size and Na(+)/K(+)-ATPase activity and protein expression after seawater exposure. Cortisol did not modify liver metabolism, but significantly suppressed gill metabolic capacity in seawater. Overall, cortisol adapts tilapia to a hyperosmotic shock associated with abrupt seawater exposure. This involves upregulation of OSTF1 gene expression and a concomitant suppression of branchial metabolism in tilapia.

Effect of moderate hypoxia at three acclimation temperatures on stress responses in Atlantic cod with different haemoglobin types.

This study examines stress responses in Atlantic cod (Gadus morhua) when exposed to a moderate and transient reduction (35% O(2) sat.) in dissolved oxygen at a range of temperatures (5 degrees C, 10 degrees C and 15 degrees C), conditions occurring in some areas they inhabit. Given their geographical distribution pattern, and differences in preferred temperature of cod with different haemoglobin types, the study was extended to include haemoglobin polymorphism. We hypothesised that the differences in temperature preference between HbI-1 and HbI-2 type cod might also be reflected in a difference in stress response to hypoxia exposure. Two hsp70-isoforms (labelled a and b) were detected and they differed in expression in the gills but not in the liver of Atlantic cod. Acclimation temperature significantly affected the expression of hsp70 in the liver, and in an isoform-specific manner in the gills. Hypoxia exposure increased the expression of hsp70 in the liver, but not the gills, of cod and this response was not influenced by the acclimation temperature. The expression of hsp70 in both tissues did not differ between fish with different haemoglobin types. Acclimation temperature significantly impacted plasma cortisol but not lactate levels. Also, acute oxygen limitation or HbI-type significantly elevated plasma cortisol and lactate levels but these responses were not modulated by acclimation temperature. Taken together, our results suggest that both temperature acclimation and acute hypoxic exposure influence the organismal and cellular stress responses in Atlantic cod. We hypothesise that HbI-2 fish are more tolerant to short-term hypoxic episodes than HbI-1 fish, and this adaptation may be independent of tissue hsp70 expression.

Effect of cortisol on melatonin production by the pineal organ of tilapia, Oreochromis mossambicus.

The aim of the present study was to examine the involvement of cortisol on melatonin synthesis in the pineal organ of the Mozambique tilapia, Oreochromis mossambicus. The circulating levels of melatonin in this species exhibited daily variations with a decrease during the photophase (0600, 1200, and 1800 h) and an increase during the scotophase (0000 h), while cortisol levels peaked during the early photophase (0600 h). The pineal organ was cultured in vitro in the dark in the presence of cortisol mimicking either stressed (100 ng/mL) or resting (10 ng/mL) concentration in tilapia. High cortisol concentration significantly reduced the levels of melatonin secreted into the medium. In the fish reared under stressful conditions, the nocturnal circulating levels of cortisol increased significantly, while melatonin did not change significantly. We detected glucocorticoid receptor (GR) transcripts in the pineal organ and a quantitative real-time PCR revealed that this receptor mRNA abundance fluctuated diurnally, increasing at 0600, 1800, and 0000 h and decreasing at 1200 h. The GR mRNA abundance in the pineal organ was not altered either in vitro when the organ was cultured in the presence of 100 ng/mL cortisol or in vivo when the fish were reared under stressful conditions. On the basis of these findings, it is proposed that cortisol lowers melatonin synthesis in the pineal organ, while the role of GR signaling in this process remains to be established.

Developmental Neurotoxicity of the Harmful Algal Bloom Toxin Domoic Acid: Cellular and Molecular Mechanisms Underlying Altered Behavior in the Zebrafish Model.

BACKGROUND: Harmful algal blooms (HABs) produce potent neurotoxins that threaten human health, but current regulations may not be protective of sensitive populations. Early life exposure to low levels of the HAB toxin domoic acid (DomA) produces long-lasting behavioral deficits in rodent and primate models; however, the mechanisms involved are unknown. The zebrafish is a powerful in vivo vertebrate model system for exploring cellular processes during development and thus may help to elucidate mechanisms of DomA developmental neurotoxicity. OBJECTIVES: We used the zebrafish model to investigate how low doses of DomA affect the developing nervous system, including windows of susceptibility to DomA exposure, structural and molecular changes in the nervous system, and the link to behavioral alterations. METHODS: To identify potential windows of susceptibility, DomA (0.09-0.18 ng) was delivered to zebrafish through caudal vein microinjection during distinct periods in early neurodevelopment. Following exposure, structural and molecular targets were identified using live imaging of transgenic fish and RNA sequencing. To assess the functional consequences of exposures, we quantified startle behavior in response to acoustic/vibrational stimuli. RESULTS: Larvae exposed to DomA at 2 d postfertilization (dpf), but not at 1 or 4 dpf, showed consistent deficits in startle behavior at 7 dpf, including lower responsiveness and altered kinematics. Similarly, myelination in the spinal cord was disorganized after exposure at 2 dpf but not 1 or 4 dpf. Time-lapse imaging revealed disruption of the initial stages of myelination. DomA exposure at 2 dpf down-regulated genes required for maintaining myelin structure and the axonal cytoskeleton. DISCUSSION: These results in zebrafish reveal a developmental window of susceptibility to DomA-induced behavioral deficits and identify altered gene expression and disrupted myelin structure as possible mechanisms. The results establish a zebrafish model for investigating the mechanisms of developmental DomA toxicity, including effects with potential relevance to exposed sensitive human populations. https://doi.org/10.1289/EHP6652.

Developmental Exposure to PCB153 (2,2',4,4',5,5'-Hexachlorobiphenyl) Alters Circadian Rhythms and the Expression of Clock and Metabolic Genes.

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.

Stress transcriptomics in fish: a role for genomic cortisol signaling.

The physiological responses to stressors, including hormonal profiles and associated tissue responsiveness have been extensively studied in teleosts, but the molecular mechanisms associated with this adaptive response are not well understood. The advent of cDNA microarray technology has transformed the field of functional genomics by revealing global gene expression changes in response to stressor exposures even in non-mammalian vertebrates, including fish. A unifying response in studies related to stressor exposure is activation of the hypothalamus-pituitary-interrenal (HPI) axis in fish, leading to cortisol release into the circulation. Here we will discuss the implications of some of the gene expression changes observed in response to acute stress in fish, while highlighting a role for cortisol in this adaptive stress response. As liver is a key organ for metabolic adjustments to stressors and also is a major target for cortisol action, the genomic studies pertaining to stress and glucocorticoid regulation have focused mainly on this tissue. The studies have identified several genes that are altered transiently after an acute stressor exposure in fish. A number of these stress-responsive genes were also modulated by glucocorticoid receptor activation, suggesting that elevation in cortisol levels during stressor exposure may be a key signal for target tissue molecular programming, essential for stress adaptation. The identification of regulatory gene networks that are stress activated, and modulated by cortisol, both in hepatic and extra-hepatic tissues, including gonads, brain, immune cells and gills, will provide a mechanistic framework to characterize the multifaceted role of cortisol during stress adaptation.

Evolutionary transition between invertebrates and vertebrates via methylation reprogramming in embryogenesis.

Major evolutionary transitions are enigmas, and the most notable enigma is between invertebrates and vertebrates, with numerous spectacular innovations. To search for the molecular connections involved, we asked whether global epigenetic changes may offer a clue by surveying the inheritance and reprogramming of parental DNA methylation across metazoans. We focused on gametes and early embryos, where the methylomes are known to evolve divergently between fish and mammals. Here, we find that methylome reprogramming during embryogenesis occurs neither in pre-bilaterians such as cnidarians nor in protostomes such as insects, but clearly presents in deuterostomes such as echinoderms and invertebrate chordates, and then becomes more evident in vertebrates. Functional association analysis suggests that DNA methylation reprogramming is associated with development, reproduction and adaptive immunity for vertebrates, but not for invertebrates. Interestingly, the single HOX cluster of invertebrates maintains unmethylated status in all stages examined. In contrast, the multiple HOX clusters show dramatic dynamics of DNA methylation during vertebrate embryogenesis. Notably, the methylation dynamics of HOX clusters are associated with their spatiotemporal expression in mammals. Our study reveals that DNA methylation reprogramming has evolved dramatically during animal evolution, especially after the evolutionary transitions from invertebrates to vertebrates, and then to mammals.

Molecular characterization, tissue-specific expression, and regulation of melanocortin 2 receptor in rainbow trout.

ACTH, the primary secretagogue for corticosteroid biosynthesis, binds to melanocortin 2 receptor (MC2R) and activates the signaling cascade leading to steroid biosynthesis in the adrenal cortex. Whereas MC2R regulation has been studied using mammalian models, little is known about the molecular mechanisms involved in ACTH signaling in nonmammalian vertebrates. A full-length cDNA encoding MC2R was sequenced from rainbow trout (Oncorhynchus mykiss) interrenal tissue (analogous to the adrenal cortex in mammals) and showed about 60 and about 44% amino acid sequence similarity to teleosts and humans, respectively. Phylogenetic analysis confirmed that MC2R from all species clustered together and was distant from other MCRs. Quantitative real-time PCR revealed a marked tissue-specific difference in MC2R mRNA abundance, with the highest levels observed in the interrenal tissue, ovary, and testis. Acute ACTH, but not alpha-MSH or [Nle4, d-Phe7]-MSH, stimulation resulted in a time- and dose-related elevation in MC2R mRNA abundance in the interrenal tissue. This corresponded with higher steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage enzyme gene expression as well as elevated cortisol production. An acute stressor transiently elevated plasma ACTH and cortisol levels at 1 h, and this was followed by a significant increase in MC2R mRNA abundance at 4 h after stressor exposure. Taken together, our results demonstrate that ACTH regulation of MC2R is highly conserved in vertebrates, whereas the tissue-specific distribution of this receptor transcript level leads us to propose a role for ACTH signaling in the stressor-mediated suppression of sex steroid levels in fish.