6PPD and its metabolite 6PPDQ induce different developmental toxicities and phenotypes in embryonic zebrafish.

The automobile tire antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its quinone metabolite 6PPDQ have recently received much attention for their acute aquatic toxicity. The present study investigated the mechanistic developmental toxicity of 6PPD and 6PPDQ in embryonic zebrafish. Neither compound induced significant mortality but significantly decreased spontaneous embryo movement and heart rate. Both compounds induced malformations with different phenotypes; the 6PPD-exposed larvae manifested a myopia-like phenotype with a convex eyeball and fusion vessels, while the 6PPDQ-exposed embryonic zebrafish manifested enlarged intestine and blood-coagulated gut, activated neutrophils, and overexpressed enteric neurons. mRNA-Seq and quantitative real-time PCR assays showed that 6PPD- and 6PPDQ-induced distinct differential gene expression aligned with their toxic phenotype. 6PPD activated the retinoic acid metabolic gene cyp26a, but 6PPDQ activated adaptive cellular response to xenobiotics gene cyp1a. 6PPD suppressed the gene expression of the eye involved in retinoic acid metabolism, phototransduction, photoreceptor function and visual perception. In contrast, 6PPDQ perturbed genes involved in inward rectifier K+ and voltage-gated ion channels activities, K+ import across the plasma membrane, iron ion binding, and intestinal immune network for IgA production. The current study advances the present understanding the reason of why many fish species are so adversely impacted by 6PPD and 6PPDQ.

Zn-Al layered double hydroxides induce embryo malformations and impair locomotion behavior in Danio rerio.

Layered double hydroxides (LDHs) are stimuli-responsive anionic nanoclays. The vast possibilities of using LDHs can lead to their existence in the ecosystem, raising a question of potential ecological concern. However, little is known about the effect of these nanomaterials on freshwater organisms. The present study aimed to assess the ecotoxicological effects of Zinc-Aluminium LDH-nitrate (ZnAl LDH-NO3) in zebrafish (Danio rerio) early life stages. The endpoints measured were mortality, malformations and hatching rate after exposure of D. rerio embryos and larvae to ZnAl LDH-NO3 following the OECD 236 guideline. The behavioral, biochemical (markers of oxidative stress and neurotoxicity), and molecular (at DNA level) alterations were also assessed using sub-lethal concentrations. No observable acute effects were detected up to 415.2 mg LDH/L while the 96 h-LC50 was estimated as 559.9 mg/L. Tested LDH caused malformations in D. rerio embryos, such as pericardial edema, incomplete yolk sac absorption and tail deformities (96 h-EC50 = 172.4 mg/L). During the dark periods, the locomotor behavior in zebrafish larvae was affected upon ZnAl LDH-NO3 exposure. However, no significant biochemical and molecular changes were recorded. The present findings suggest that ZnAl LDH-NO3 can be regarded as a non-toxic nanomaterial towards D. rerio (E/LC50 > > 100 mg/L) although impairment of the locomotion behavior on zebrafish embryos can be expected at concentrations below 100 mg/L.

Environmental exposure to triazole fungicide causes left-right asymmetry defects and contributes to abnormal heart development in zebrafish embryos by activating PPARγ-coupled Wnt/ß-catenin signaling pathway.

Triazole fungicides have been widely used all over the world. However, their potential ecological safety and health risks remain unclear, especially their cardiac developmental toxicity. This study systematically investigated whether and how triazole fungicides could activate peroxisome proliferative activity receptor γ (PPARγ) to cause abnormal heart development. Among ten triazole fungicides, difenoconazole (DIF) exhibited the strongest agonistic activity and caused severe pericardial edema in zebrafish embryos, accompanied by a reduction in heart rate, blood flow and cardiac function. In vitro transcriptomic profile implicated that DIF inhibited the Wnt signaling pathway, and in vivo DIF exposure significantly increased the phosphorylation of ß-catenin (p = 0.0002) and altered the expression of related genes in zebrafish embryos. Importantly, exposure to DIF could activate PPARγ and inhibit the Wnt/ß-catenin signaling pathway, which changed the size of Kupffer's vesicle (KV) (p = 0.02), altered the expression of left-right (LR) asymmetry-related genes, caused cardiac LR asymmetry defect, and eventually led to abnormal heart development. These findings provide evidence for potential developmental toxicity of triazole fungicides and highlight the necessity of assessing their ecological safety and human health risks.

Molecular and behavioral assessment in larval zebrafish (Danio rerio) following exposure to environmentally relevant levels of the antineoplastic cyclophosphamide.

Antineoplastics treat cancers and enter aquatic ecosystems through wastewater and hospital effluent. Risks associated with antineoplastics are not well characterized in aquatic organisms. We conducted zebrafish embryo/larvae toxicity assays to evaluate responses to cyclophosphamide (0.01-50 µM). Zebrafish survival was affected by 5 µM cyclophosphamide and deformities were noted at > 1 µM. Oxidative respiration remained unchanged in embryos with exposure up to 200 µM. Reactive oxygen species were not increased by 50 µM cyclophosphamide exposure. More than 15 oxidative stress and immune-related transcripts were measured. Superoxide dismutase 2 and heat shock protein 70 and 90a were induced in larvae by cyclophosphamide. Immune-related transcripts were assessed due to immunosuppressive properties of cyclophosphamide, and mmp9 and myd88 levels were altered in expression. Hyperactivity of larvae was noted following 5 µM cyclophosphamide exposure. There was no change in anxiety-related endpoints (light-dark preference). Risks for larval fish exposed to cyclophosphamide in the environment may be low.

Cadmium significantly changes major morphometrical points and cardiovascular functional parameters during early development of zebrafish.

Living organisms are commonly exposed to cadmium and other toxic metals. A vast body of research has shown the significant effects of these toxic metals on developmental processes. In order to study the role of toxic metals on early developmental stages of eukaryotes, we explored the effect of cadmium (Cd2+) contaminant on zebrafish. Thus, zebrafish embryos were exposed to 3 mg/L (16.7 µM) Cd2+ for 96 h and imaged every 24 h from the exposure onwards. Hatching rates of the eggs were determined at 72 h, followed by analyses at 96 h for: survival rate, morphometrical factors, and functional parameters of the cardiovascular system. Interestingly enough, significant hatching delays along with smaller cephalic region and some morphological abnormalities were observed in the treatment group. Moreover, substantial changes were noticed in the length of notochord and embryo, absorption of yolk sac with shorter extension, area of swimming bladder, as well as pericardium sac after Cd2+ treatment. Cadmium also caused significant abnormalities in heart physiology which could be the leading cause of mentioned morphological deformities. Herein, our results shine light on systematic acute embryological effects of cadmium in the early development of zebrafish for the first time.

Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Short exposure to glyphosate induces locomotor, craniofacial, and bone disorders in zebrafish (Danio rerio) embryos.

Glyphosate [N-(phosphonomethyl)glycine] is the active ingredient in widely used broad-spectrum herbicides. Even though the toxicity mechanism of this herbicide in vertebrates is poorly understood, evidence suggests that glyphosate is an endocrine disruptor capable of producing morphological anomalies as well as cardiotoxic and neurotoxic effects. We used the zebrafish model to assess the effects of early life glyphosate exposure on the development of cartilage and bone tissues and organismal responses. We found functional alterations, including a reduction in the cardiac rate, significant changes in the spontaneous tail movement pattern, and defects in craniofacial development. These effects were concomitant with alterations in the level of the estrogen receptor alpha osteopontin and bone sialoprotein. We also found that embryos exposed to glyphosate presented spine deformities as adults. These developmental alterations are likely induced by changes in protein levels related to bone and cartilage formation.

24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos.

Previous embryonic fish data have shown caffeine to induce potential teratogenic and long-term neurodevelopmental outcomes through oxidative stress-mediated apoptosis. In this context, antioxidants may have the potential to counteract the caffeine-induced effects. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proven antioxidant properties, against caffeine-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 0.5 mM caffeine, co-exposed to 24-EPI (0.01, 0.1 and 1 µM) and to 24-EPI alone (1 µM) for 96 h. During exposure, lethal and sublethal developmental parameters were evaluated. At the end of the exposure, biochemical evaluations were made, and 24 h after, different behavioural paradigms were assessed. An increased number of animals showing oedema and malformations were observed after caffeine exposure, while these were reduced after co-exposure to 24-EPI concentration, namely the tail curvature. The results showed oxidative stress and related parameters similar among treatments. Yet, caffeine exposure resulted in locomotor deficits (decreased speed and distance) and disrupted anxiety-like and avoidance responses. The co-exposure to caffeine and to the highest 24-EPI concentrations resulted in less pronounced behavioural deficits. Overall, there was an absence of effects in the embryo/larvae exposed solely to 24-EPI, while caffeine caused developmental and neurotoxic effects. Although further studies are needed, the results showed promising protective effects of the highest 24-EPI concentration tested against the toxicity induced by caffeine in zebrafish.

Modulation of PPAR signaling disrupts pancreas development in the zebrafish, Danio rerio.

Peroxisome Proliferator Activated Receptors (PPARs) are transcription factors that regulate processes such as lipid and glucose metabolism. Synthetic PPAR ligands, designed as therapeutics for metabolic disease, provide a tool to assess the relationship between PPAR activity and pancreas development in vivo, an area that remains poorly characterized. Here, we aim to assess the effects of PPAR agonists and antagonists on gene expression, embryonic morphology and pancreas development in transgenic zebrafish embryos. To evaluate developmental perturbations, we assessed gross body and pancreas morphology at 4 days post fertilization (dpf) in response to developmental exposures with PPARα, PPARγ, and PPARß/δ agonists and antagonists at 0, 0.01, 0.1, 1, and 10 µM concentrations. All ligand exposures, with the exception of the PPARα agonist, resulted in significantly altered fish length and yolk sac area. PPARγ agonist and antagonist had higher incidence of darkened yolk sac and craniofacial deformities, whereas PPARα antagonist had higher incidence of pericardial edema and death. Significantly reduced endocrine pancreas area was observed in both PPARγ ligands and PPARα agonist exposed embryos, some of which also exhibited aberrant endocrine pancreas morphology. Both PPARß/δ ligands caused reduced exocrine pancreas length and novel aberrant phenotype, and disrupted gene expression of pancreatic targets pdx1, gcga, and try. Lipid staining was performed at 8 dpf and revealed altered lipid accumulation consistent with isoform function. These data indicate chronic exposure to synthetic ligands may induce morphological and pancreatic defects in zebrafish embryos.

Developmental phenotypic and transcriptomic effects of exposure to nanomolar levels of metformin in zebrafish.

Metformin is found in the majority of lakes and streams in the United States, leading to widespread environmental exposure. Results of the present study indicate that extended duration metformin exposure at critical developmental periods leads to decreased survival rates in zebrafish (danio rerio), an NIH approved human model. Significant abnormalities are seen with extended duration metformin exposure from 4 h post fertilization up to 5 days post fertilization, although short term metformin exposure for 24 h at 4-5 days post fertilization did not lead to any significant abnormalities. Both extended and short term duration did however have an impact on locomotor activity of zebrafish, and several genes involved in neurological and cardiovascular development were differentially expressed after exposure to metformin. The changes seen in behavior, gene expression and morphological abnormalities caused by metformin exposure should be examined further in future studies in order to assess their potential human health implications as metformin prescriptions continue to increase worldwide.

Radioprotective Effects of Kelulut Honey in Zebrafish Model.

Large doses of ionizing radiation can damage human tissues. Therefore, there is a need to investigate the radiation effects as well as identify effective and non-toxic radioprotectors. This study evaluated the radioprotective effects of Kelulut honey (KH) from stingless bee (Trigona sp.) on zebrafish (Danio rerio) embryos. Viable zebrafish embryos at 24 hpf were dechorionated and divided into four groups, namely untreated and non-irradiated, untreated and irradiated, KH pre-treatment and amifostine pre-treatment. The embryos were first treated with KH (8 mg/mL) or amifostine (4 mM) before irradiation at doses of 11 Gy to 20 Gy using gamma ray source, caesium-137 (137Cs). Lethality and abnormality analysis were performed on all of the embryos in the study. Immunohistochemistry assay was also performed using selected proteins, namely γ-H2AX and caspase-3, to investigate DNA damages and incidences of apoptosis. KH was found to reduce coagulation effects at up to 20 Gy in the lethality analysis. The embryos developed combinations of abnormality, namely microphthalmia (M), body curvature and microphthalmia (BM), body curvature with microphthalmia and microcephaly (BMC), microphthalmia and pericardial oedema (MO), pericardial oedema (O), microphthalmia with microcephaly and pericardial oedema (MCO) and all of the abnormalities (AA). There were more abnormalities developed from 24 to 72 h (h) post-irradiation in all groups. At 96 h post-irradiation, KH was identified to reduce body curvature effect in the irradiated embryos (up to 16 Gy). γ-H2AX and caspase-3 intensities in the embryos pre-treated with KH were also found to be lower than the untreated group at gamma irradiation doses of 11 Gy to 20 Gy and 11 Gy to 19 Gy, respectively. KH was proven to increase the survival rate of zebrafish embryos and exhibited protection against organ-specific abnormality. KH was also found to possess cellular protective mechanism by reducing DNA damage and apoptosis proteins expression.

L-Carnitine ameliorates congenital myopathy in a tropomyosin 3 de novo mutation transgenic zebrafish.

BACKGROUND: Congenital myopathy (CM) is a group of clinically and genetically heterogeneous muscle disorders, characterized by muscle weakness and hypotonia from birth. Currently, no definite treatment exists for CM. A de novo mutation in Tropomyosin 3-TPM3(E151G) was identified from a boy diagnosed with CM, previously TPM3(E151A) was reported to cause CM. However, the role of TPM3(E151G) in CM is unknown. METHODS: Histopathological, swimming behavior, and muscle endurance were monitored in TPM3 wild-type and mutant transgenic fish, modelling CM. Gene expression profiling of muscle of the transgenic fish were studied through RNAseq, and mitochondria respiration was investigated. RESULTS: While TPM3(WT) and TPM3(E151A) fish show normal appearance, amazingly a few TPM3(E151G) fish display either no tail, a crooked body in both F0 and F1 adults. Using histochemical staining for the muscle biopsy, we found TPM3(E151G) displays congenital fiber type disproportion and TPM3(E151A) resembles nemaline myopathy. TPM3(E151G) transgenic fish dramatically swimming slower than those in TPM3(WT) and TPM3(E151A) fish measured by DanioVision and T-maze, and exhibit weaker muscle endurance by swimming tunnel instrument. Interestingly, L-carnitine treatment on TPM3(E151G) transgenic larvae significantly improves the muscle endurance by restoring the basal respiration and ATP levels in mitochondria. With RNAseq transcriptomic analysis of the expression profiling from the muscle specimens, it surprisingly discloses large downregulation of genes involved in pathways of sodium, potassium, and calcium channels, which can be rescued by L-carnitine treatment, fatty acid metabolism was differentially dysregulated in TPM3(E151G) fish and rescued by L-carnitine treatment. CONCLUSIONS: These results demonstrate that TPM3(E151G) and TPM3(E151A) exhibit different pathogenicity, also have distinct gene regulatory profiles but the ion channels were downregulated in both mutants, and provides a potential mechanism of action of TPM3 pathophysiology. Our results shed a new light in the future development of potential treatment for TPM3-related CM.

Evaluation of the potential environmental risk from the destination of medicines: an epidemiological and toxicological study.

BACKGROUND: The high consumption of medicines by the population and their storage at home might cause an increase in the number of pharmaceutical substances that may be inappropriately discarded in the sanitary sewage, reaching an environmental aquatic. Thus, the effects of these emerging contaminants need more studies. OBJECTIVES: To identify the profile of most medicines that are discarded by users of community pharmacy and evaluate the toxicity of the most disposed drugs. METHODS: This was a translational study. A descriptive observational study was carried out for convenience of community pharmacy users using a standardized questionnaire. Subsequently, the lethal concentration 50 (LC50) for medicine that is most frequently discarded was determined. After LC50, the embryos (n = 144) were exposed to sublethal concentrations for most discarded drug at 24, 48, and 72 h. Mortality, heartbeat, and embryo deformities were used as parameters of toxicity. RESULTS: Most respondents (96%) had a "home pharmacy." The primary forms of disposal were in the common household waste, kitchen sink, and/or bathroom. The medicines that were most incorrectly discarded by the interviewees were nimesulide (17.1%), dipyrone (10.7%), and paracetamol (5.2%). LC50 of nimesulide was calculated (0.92 µgmL-1). The toxicological test revealed that embryos exposed to nimesulide showed several abnormalities, such as defects in the spinal cord, tail, yolk sac, as well as pericardial edema. Furthermore, the heartbeat decreased by 30% at a concentration of 0.4 µgmL-1 as compared with control group. The yolk sac and pericardial areas increased to >100% in all treatment groups when compared with the control group. CONCLUSION: Respondents disposed medicines in an inappropriate manner primarily in household waste and in the toilet. Nimesulide was the most discarded drug according to study population. Moreover, teratogenic effects such as spinal cord defects, decreasing heartbeats, and increasing pericardial and yolk sac area in embryos were observed after exposure to nimesulide. These results show that nimesulide may promote risk to aquatic organisms and to human health if it is discarded in an unsafe manner.

Assessment of the in vitro developmental toxicity of diethylstilbestrol and estradiol in the zebrafish embryotoxicity test.

The present study investigated the developmental toxicity of diethylstilbestrol (DES) in the zebrafish embryotoxicity test (ZET). This was done to investigate whether the ZET would better capture the developmental toxicity of DES than the embryonic stem cells test (EST) that was previously shown to underpredict the DES-induced developmental toxicity as compared to in vivo data, potentially because the EST does not capture late events in the developmental process. The ZET results showed DES-induced growth retardation, cumulative mortality and dysmorphisms (i.e. induction of pericardial edema) in zebrafish embryos while the endogenous ERα agonist 17ß-estradiol (E2) showed only growth retardation and cumulative mortality with lower potency compared to DES. Furthermore, the DES-induced pericardial edema formation in zebrafish embryos could be counteracted by co-exposure with ERα antagonist fulvestrant, indicating that the ZET captures the role of ERα in the mode of action underlying the developmental toxicity of DES. Altogether, it is concluded that the ZET differentiates DES from E2 with respect to their developmental toxicity effects, while confirming the role of ERα in mediating the developmental toxicity of DES. Furthermore, comparison to in vivo data revealed that, like the EST, in a quantitative way also the ZET did not capture the relatively high in vivo potency of DES as a developmental toxicant.

Thyroid Hormone Receptor α Mutations Cause Heart Defects in Zebrafish.

Background: Mutations of thyroid hormone receptor α1 (TRα1) cause resistance to thyroid hormone (RTHα). Patients exhibit growth retardation, delayed bone development, anemia, and bradycardia. By using mouse models of RTHα, much has been learned about the molecular actions of TRα1 mutants that underlie these abnormalities in adults. Using zebrafish models of RTHα that we have recently created, we aimed to understand how TRα1 mutants affect the heart function during this period. Methods: In contrast to human and mice, the thra gene is duplicated, thraa and thrab, in zebrafish. Using CRISPR/Cas9-mediated targeted mutagenesis, we created C-terminal mutations in each of two duplicated thra genes in zebrafish (thraa 8-bp insertion or thrab 1-bp insertion mutations). We recently showed that these mutant fish faithfully recapitulated growth retardation as found in patients and thra mutant mice. In the present study, we used histological analysis, gene expression profiles, confocal fluorescence, and transmission electron microscopy (TEM) to comprehensively analyze the phenotypic characteristics of mutant fish heart during development. Results: We found both a dilated atrium and an abnormally shaped ventricle in adult mutant fish. The retention of red blood cells in the two abnormal heart chambers, and the decreased circulating blood speed and reduced expression of contractile genes indicated weakened contractility in the heart of mutant fish. These abnormalities were detected in mutant fish as early as 35 days postfertilization (juveniles). Furthermore, the expression of genes associated with the sarcomere assembly was suppressed in the heart of mutant fish, resulting in abnormalities of sarcomere organization as revealed by TEM, suggesting that the abnormal sarcomere organization could underlie the bradycardia exhibited in mutant fish. Conclusions: Using a zebrafish model of RTHα, the present study demonstrated for the first time that TRα1 mutants could act to cause abnormal heart structure, weaken contractility, and disrupt sarcomere organization that affect heart functions. These findings provide new insights into the bradycardia found in RTHα patients.

Toxicological assessment and developmental abnormalities induced by butylparaben and ethylparaben exposure in zebrafish early-life stages.

Toxicological effects of butylparaben (BuP) and ethylparaben (EtP) on zebrafish (Danio rerio) early-life stages are not well established. The present study evaluated, using zebrafish embryos and larvae, the toxicity of BuP and EtP through benchmark dose (BMD) approach. BuP was more toxic than EtP to zebrafish larvae. In fact, Lethal Concentration 50 (LC50) values at 96 h post-fertilization (hpf) for BuP and EtP were 2.34 mg/L and 20.86 mg/L, respectively. Indeed, BMD confidence interval (lower bound (BMDL) - upper bound (BMDU) was 0.91-1.92 mg/L for BuP and 10.8-17.4 mg/L for EtP. Zebrafish embryos exposed to 1 mg/L, 2.5 mg/L of BuP and 5 mg/L, 10 mg/L, 20 mg/L, 30 mg/L of EtP showed several developmental abnormalities and teratological effects compared to negative control. Exposed zebrafish developed reduced heartbeat, reduction in blood circulation, blood stasis, pericardial edema, deformed notochord and misshaped yolk sac. Embryos exposed to the highest concentrations of the chemicals (2.5 mg/L of BuP, 10 mg/L, 20 mg/L and 30 mg/L of EtP) showed the developmental abnormalities at 48 hpf while those treated with 1 mg/L of BuP and 10 mg/L of EtP reported behavioral changes at 72 hpf, including trembling of head, pectoral fins and spinal cord. This research identified the lethal and sublethal effects of BuP and EtP in zebrafish early-life stages and could be helpful to elucidate the developmental pathways of toxicity of parabens.

Multifactorial Genetic and Environmental Hedgehog Pathway Disruption Sensitizes Embryos to Alcohol-Induced Craniofacial Defects.

BACKGROUND: Prenatal alcohol exposure (PAE) is perhaps the most common environmental cause of human birth defects. These exposures cause a range of structural and neurological defects, including facial dysmorphologies, collectively known as fetal alcohol spectrum disorders (FASD). While PAE causes FASD, phenotypic outcomes vary widely. It is thought that multifactorial genetic and environmental interactions modify the effects of PAE. However, little is known of the nature of these modifiers. Disruption of the Hedgehog (Hh) signaling pathway has been suggested as a modifier of ethanol teratogenicity. In addition to regulating the morphogenesis of craniofacial tissues commonly disrupted in FASD, a core member of the Hh pathway, Smoothened, is susceptible to modulation by structurally diverse chemicals. These include environmentally prevalent teratogens like piperonyl butoxide (PBO), a synergist found in thousands of pesticide formulations. METHODS: Here, we characterize multifactorial genetic and environmental interactions using a zebrafish model of craniofacial development. RESULTS: We show that loss of a single allele of shha sensitized embryos to both alcohol- and PBO-induced facial defects. Co-exposure of PBO and alcohol synergized to cause more frequent and severe defects. The effects of this co-exposure were even more profound in the genetically susceptible shha heterozygotes. CONCLUSIONS: Together, these findings shed light on the multifactorial basis of alcohol-induced craniofacial defects. In addition to further implicating genetic disruption of the Hh pathway in alcohol teratogenicity, our findings suggest that co-exposure to environmental chemicals that perturb Hh signaling may be important variables in FASD and related craniofacial disorders.

Prostaglandins prevent acetaminophen induced embryo toxicity in zebrafish (Danio rerio).

Previous research in our laboratory showed that acetaminophen (ACE) induced embryonic mortality and abnormalities in zebrafish. Here, we examined the dose response of ACE (0.05-50 µg L-1) in zebrafish embryos. Concentrations as low as 0.1 µg L-1 significantly increased abnormalities, and all test concentrations significantly increased mortality rates. In mammals, ACE inhibits cyclooxygenase (COX) enzymes to decrease prostaglandin production. Here we report COX activity and expression of the cox-1, cox-2a, and cox-2b genes in zebrafish embryos. COX activity was significantly inhibited by specific mammalian cox-1 (SC-560) and cox-2 (DuP-697) inhibitors in unexposed and ACE-exposed embryos. COX activity declined with development time. Maternal transcripts of all cox genes were found at 1 -h post fertilization and embryonic expression began in gastrulation or early segmentation. Co-exposure of ACE and prostaglandin E2 abolished the ACE-induced effects. This strongly supports that ACE elicits embryo toxicity in zebrafish though the same molecular mechanism of action of their therapeutic effects in mammals.

Assessing the toxicity of the benzamide fungicide zoxamide in zebrafish (Danio rerio): Towards an adverse outcome pathway for beta-tubulin inhibitors.

Commercial benzamide fungicides are applied to crops to control damage caused by oomycete fungi and are used as veterinary pharmaceuticals in aquaculture. The mechanism of action of these fungicides is to induce mitotic arrest via binding to beta-tubulin, thus inhibiting tubulin polymerization. However, there are little toxicity data available for benzimidazole fungicides in fish. To address this knowledge gap, we conducted zebrafish embryo toxicity tests to assess deformities, survival, and sub-lethal responses following exposure to zoxamide (0, 0.5, 1.0, 2.5, 5.0 and 10 µM zoxamide). We hypothesized that skeletal deformities would be prevalent in zebrafish due to its mechanism of inhibiting beta-tubulin polymerization. Zoxamide was relatively toxic to zebrafish embryos and larvae, and survival was reduced ∼50 % at 2 days post fertilization (dpf) with 10 µM exposure and over time at 6 dpf, 2.5 µM exposure reduced survival by ∼20 %. Frequency of hatch was also reduced/delayed in zebrafish at 3 dpf with >2.5 µM zoxamide. Pericardial edema, body length shortening, and spine curvature were observed in larvae exposed to >5 µM. Mitochondrial bioenergetics were assessed in ∼30 hpf embryos (24-hour exposure) using an XFe24 Flux Analyzer and regression analysis revealed a negative relationship between basal respiration and zoxamide concentration. Superoxide dismutase 1 and caspase 3 mRNA levels were both decreased in 6 dpf larvae exposed to 2.5 µM zoxamide, but were not changed in expression at 0.5 nor 1 µM zoxamide. Continuous 6-day exposure to zoxamide reduced larval activity at 2.5 µM; conversely a 24-hour exposure (at 5-6 dpf) induced hyperactivity at 5 µM suggesting dose and time dependent effects on fish behavior. Based on sub-lethal endpoints, we conceptualize an adverse outcome pathway for chemicals that inhibit tubulin polymerization.

SCO-Spondin Defects and Neuroinflammation Are Conserved Mechanisms Driving Spinal Deformity across Genetic Models of Idiopathic Scoliosis.

Adolescent idiopathic scoliosis (AIS) affects 3% to 4% of children between the ages of 11 and 18 [1, 2]. This disorder, characterized by abnormal three-dimensional spinal curvatures that typically develop during periods of rapid growth, occurs in the absence of congenital vertebral malformations or neuromuscular defects [1]. Genetic heterogeneity [3] and a historical lack of appropriate animal models [4] have confounded basic understanding of AIS biology; thus, treatment options remain limited [5, 6]. Recently, genetic studies using zebrafish have linked idiopathic-like scoliosis to irregularities in motile cilia-mediated cerebrospinal fluid flow [7-9]. However, because loss of cilia motility in human primary ciliary dyskinesia patients is not fully associated with scoliosis [10, 11], other pathogenic mechanisms remain to be determined. Here, we demonstrate that zebrafish scospondin (sspo) mutants develop late-onset idiopathic-like spinal curvatures in the absence of obvious cilia motility defects. Sspo is a large secreted glycoprotein functionally associated with the subcommissural organ and Reissner's fiber [12]-ancient and enigmatic organs of the brain ventricular system reported to govern cerebrospinal fluid homeostasis [13, 14], neurogenesis [12, 15-18], and embryonic morphogenesis [19]. We demonstrate that irregular deposition of Sspo within brain ventricles is associated with idiopathic-like scoliosis across diverse genetic models. Furthermore, Sspo defects are sufficient to induce oxidative stress and neuroinflammatory responses implicated in AIS pathogenesis [9]. Through screening for chemical suppressors of sspo mutant phenotypes, we also identify potent agents capable of blocking severe juvenile spine deformity. Our work thus defines a new preclinical model of AIS and provides tools to realize novel therapeutic strategies.