Altered Larval Yellow Perch Swimming Behavior Due to Methylmercury and PCB126 Detected Using Hidden Markov Chain Models.

Fish swimming behavior is a commonly measured response in aquatic ecotoxicology because behavior is considered a whole organism-level effect that integrates many sensory systems. Recent advancements in animal behavior models, such as hidden Markov chain models (HMM), suggest an improved analytical approach for toxicology. Using both new and traditional approaches, we examined the sublethal effects of PCB126 and methylmercury on yellow perch (YP) larvae (Perca flavescens) using three doses. Both approaches indicate larvae increase activity after exposure to either chemical. The middle methylmercury-dosed larvae showed multiple altered behavior patterns. First, larvae had a general increase in activity, typically performing more behavior states, more time swimming, and more swimming bouts per second. Second, when larvae were in a slow or medium swimming state, these larvae tended to switch between these states more often. Third, larvae swam slower during the swimming bouts. The upper PCB126-dosed larvae exhibited a higher proportion and a fast swimming state, but the total time spent swimming fast decreased. The middle PCB126-dosed larvae transitioned from fast to slow swimming states less often than the control larvae. These results indicate that developmental exposure to very low doses of these neurotoxicants alters YP larvae overall swimming behaviors, suggesting neurodevelopment alteration.

Parental Whole Life Cycle Exposure to Dietary Methylmercury in Zebrafish (Danio rerio) Affects the Behavior of Offspring.

Methylmercury (MeHg) is an established neurotoxicant of concern to fish-eating organisms. While most studies have focused on the fish consumers, much less is known about the effects of MeHg on the fish themselves, especially following exposures to chronic and environmentally relevant scenarios. Here we evaluated the behavioral effects of developmental MeHg insult by exposing parental generations of zebrafish to an environmentally realistic MeHg dietary concentration (1 ppm) and two higher concentrations (3 and 10 ppm) throughout their whole life span. Upon reaching adulthood, their offspring were analyzed through a series of behavioral tests, including the visual-motor response (VMR) assay, analysis of spontaneous swimming and evaluation of foraging efficiency. The VMR assay identified decreased locomotor output in the 6 day postfertilization (dpf) offspring of fish exposed to 3 and 10 ppm MeHg. However, in a second test 7 dpf fish revealed an increase in locomotor activity in all MeHg exposures tested. Increases in locomotion continued to be observed until 16 dpf, which coincided with increased foraging efficiency. These results suggest an association between MeHg and hyperactivity, and imply that fish chronically exposed to MeHg in the wild may be vulnerable to predation.

Understanding Genetics and Pediatric Cardiac Health.

UNLABELLED: Congenital heart defects (CHD) continue to be the most prevalent birth defect that occurs worldwide in approximately 6-8 of every 1,000 live births. High rates of morbidity and mortality in infants, children, and adults living with CHD place a growing need for health care professionals (HCPs) to better understand potentially modifiable genetic and environmental influences. This paper will present examples of research and governmental initiatives that support genetics education and research and a review of known genetic factors associated with CHD development. ORGANIZING CONSTRUCT: A review of the known genetic factors on risk for CHD formation in infants will be provided to help health care professionals gain a greater understanding of the genetic influences on pediatric cardiac health. CONCLUSIONS: There are known genetic pathways and risk factors that contribute to development of CHD. This paper is a primer for nurses and HCPs providing information of the genetics and inheritance patterns of CHD to be useful in daily clinical practice. CLINICAL RELEVANCE: Nurses work in multiple communities where they are uniquely positioned to educate and provide information about research and current models of care with families affected by CHD. Nurses and HCPs who better understand genetic risk factors associated with CHD development can more promptly refer and offer treatment for these children and families thus providing individuals of childbearing age with the necessary resources and information about risk factors.

Engineered nanomaterials: an emerging class of novel endocrine disruptors.

Over the past decade, engineered nanomaterials (ENMs) have garnered great attention for their potentially beneficial applications in medicine, industry, and consumer products due to their advantageous physicochemical properties and inherent size. However, studies have shown that these sophisticated molecules can initiate toxicity at the subcellular, cellular, and/or tissue/organ level in diverse experimental models. Investigators have also demonstrated that, upon exposure to ENMs, the physicochemical properties that are exploited for public benefit may mediate adverse endocrine-disrupting effects on several endpoints of mammalian reproductive physiology (e.g., steroidogenesis, spermatogenesis, pregnancy). Elucidating these complex interactions within reproductive cells and tissues will significantly advance our understanding of ENMs as an emerging class of novel endocrine disruptors and reproductive toxicants. Herein we reviewed the recent developments in reproductive nanotoxicology and identified the gaps in our knowledge that may serve as future research directions to foster continued advancement in this evolving field of study.

An evolutionarily conserved mechanism of calcium-dependent neurotoxicity in a zebrafish model of fetal alcohol spectrum disorders.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a leading cause of neurodevelopmental disability. Nonhuman animal models offer novel insights into its underlying mechanisms. Although the developing zebrafish has great promise for FASD research, a significant challenge to its wider adoption is the paucity of clear, mechanistic parallels between its ethanol (EtOH) responses and those of nonpiscine, established models. Inconsistencies in the published pharmacodynamics for EtOH-exposed zebrafish, alongside the use of comparatively high EtOH doses, challenge the interpretation of this model's clinical relevance. METHODS: To address these limitations, we developed a binge, single-exposure model of EtOH exposure in the early zebrafish embryo. RESULTS: Brief (3-hour) EtOH exposure is sufficient to cause significant neural crest losses and craniofacial alterations, with peak vulnerability during neurogenesis and early somitogenesis. These losses are apoptotic, documented using TUNEL assay and secA5-YFP-reporter fish. Apoptosis is dose dependent with an EC50 = 56.2 ± 14.3 mM EtOHint , a clinically relevant value within the range producing apoptosis in chick and mouse neural crest. This apoptosis requires the calcium-dependent activation of CaMKII and recapitulates the well-described EtOH signaling mechanism in avian neural crest. Importantly, we resolve the existing confusion regarding zebrafish EtOH kinetics. We show that steady-state EtOH concentrations within both chorion-intact and dechorionated embryos are maintained at 35.7 ± 2.8% of EtOHext levels across the range from 50 to 300 mM EtOHext , a value consistent with several published reports. Equilibrium is rapid and complete within 5 minutes of EtOH addition. CONCLUSIONS: The calcium/CaMKII mechanism of EtOH's neurotoxicity is shared between an amniote (chick) and teleost fish, indicating that this mechanism is evolutionarily conserved. Our data suggest that EtOHext concentrations >2% (v/v) for chorion-intact embryos and 1.5% (v/v) for dechorionated embryos have limited clinical relevance. The strong parallels with established models endorse the zebrafish's relevance for mechanistic studies of EtOH's developmental neurotoxicity.

Bringing Real Inquiry-Based Science to Diverse Secondary Educational Environments: A Virtual Zebrafish Laboratory to Investigate Environmental Health.

The goal of the University of Wisconsin-Milwaukee WInSTEP SEPA program is to provide valuable and relevant research experiences to students and instructors in diverse secondary educational settings. Introducing an online experience allows the expansion of a proven instructional research program to a national scale and removes many common barriers. These can include lack of access to zebrafish embryos, laboratory equipment, and modern classroom facilities, which often deny disadvantaged and underrepresented students from urban and rural school districts valuable inquiry-based learning opportunities. An online repository of zebrafish embryo imagery was developed in the Carvan laboratory to assess the effects of environmental chemicals. The WInSTEP SEPA program expanded its use as an accessible online tool, complementing the existing classroom experience of our zebrafish module. This virtual laboratory environment contains images of zebrafish embryos grown in the presence of environmental toxicants (ethanol, caffeine, and nicotine), allowing students to collect data on 19 anatomical endpoints and generate significant amounts of data related to developmental toxicology and environmental health. This virtual laboratory offers students and instructors the choice of data sets that differ in the independent variables of chemical concentration and duration of postfertilization exposure. This enables students considerable flexibility in establishing their own experimental design to match the curriculum needs of each instructor.

Differential gene expression associated with dietary methylmercury (MeHg) exposure in rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio).

The objective of this study was to identify and evaluate conserved biomarkers that could be used in most species of teleost fish at most life-stages. We investigated the effects of sublethal methylmercury (MeHg) exposure on developing rainbow trout and zebrafish. Juvenile rainbow trout and young adult zebrafish were fed food with MeHg added at 0, 0.5, 5, and 50 ppm. Atomic absorption spectrometry was applied to measure whole body total Hg levels, and pathologic analysis was performed to identify MeHg-induced toxicity. Fish at 6 weeks were sampled from each group for microarray analysis using RNA from whole fish. MeHg-exposed trout and zebrafish did not show overt signs of toxicity or pathology, nor were significant differences seen in mortality, length, mass, or condition factor. The accumulation of MeHg in trout and zebrafish exhibited dose- and time-dependent patterns during 6 weeks, and zebrafish exhibited greater assimilation of total Hg than rainbow trout. The dysregulated genes in MeHg-treated fish have multiple functional annotations, such as iron ion homeostasis, glutathione transferase activity, regulation of muscle contraction, troponin I binding and calcium-dependent protein binding. Genes were selected as biomarker candidates based on their microarray data and their expression was evaluated by QPCR. Unfortunately, these genes are not good consistent biomarkers for both rainbow trout and zebrafish from QPCR evaluation using individual fish. Our conclusion is that biomarker analysis for aquatic toxicant assessment using fish needs to be based on tissue-, sex- and species-specific consideration.

Absence of fractionation of mercury isotopes during trophic transfer of methylmercury to freshwater fish in captivity.

We performed two controlled experiments to determine the amount of mass-dependent and mass-independent fractionation (MDF and MIF) of methylmercury (MeHg) during trophic transfer into fish. In experiment 1, juvenile yellow perch (Perca flavescens) were raised in captivity on commercial food pellets and then their diet was either maintained on unamended food pellets (0.1 µg/g MeHg) or was switched to food pellets with 1.0 µg/g or 4.0 µg/g of added MeHg, for a period of 2 months. The difference in δ(202)Hg (MDF) and Δ(199)Hg (MIF) between fish tissues and food pellets with added MeHg was within the analytical uncertainty (δ(202)Hg, 0.07 ‰; Δ(199)Hg, 0.06 ‰), indicating no isotope fractionation. In experiment 2, lake trout (Salvelinus namaycush) were raised in captivity on food pellets and then shifted to a diet of bloater (Coregonus hoyi) for 6 months. The δ(202)Hg and Δ(199)Hg of the lake trout equaled the isotopic composition of the bloater after 6 months, reflecting reequilibration of the Hg isotopic composition of the fish to new food sources and a lack of isotope fractionation during trophic transfer. We suggest that the stable Hg isotope ratios in fish can be used to trace environmental sources of Hg in aquatic ecosystems.

Mitigative effects of natural and model dissolved organic matter with different functionalities on the toxicity of methylmercury in embryonic zebrafish.

Dissolved organic matter (DOM) occurs ubiquitously in aquatic environments and plays an intrinsic role in altering the chemical speciation and toxicity of methylmercury (MeHg). However, interactions between MeHg and natural DOM remain poorly understood, especially at the functional group level. We report here the mitigative effects of three natural organic matter (NOM) and five model-DOM under different concentrations (0, 1, 3, 10, 30 and 100 mg-C/L) on the toxicity of MeHg in embryonic zebrafish (<4 h post-fertilization, hpf). NOM are those from the Mississippi River, Yukon River, and Suwannee River, while model-DOM include those containing thiosalicylic acid, L-glutathione, dextran, alginic acid, and humic acid. We selected a MeHg concentration (100 n-mol/L) that reduces the survival rate of embryos at 24 hpf by 18% and increases malformations at 72 and 96 hpf. In the presence of DOM, however, the malformation rates induced by MeHg can be mitigated to a different extent depending on DOM concentrations, specific functional groups, and/or specific components. Model DOM with aromatic thiols was the most effective at mitigating the effects of MeHg, followed by L-glutathione, carbohydrates, and humic acid. NOM also mitigated the toxicity of MeHg dependent on their composition and/or effective DOM components as characterized by fluorescence excitation-emission matrix techniques. Specifically, humic-like DOM components are more effective in reducing the MeHg toxicity in the embryonic zebrafish compared to protein-like components. Further studies are needed to elucidate the interactions between DOM and MeHg and the mitigative mechanisms at the molecular level.

Selenomethionine reduces visual deficits due to developmental methylmercury exposures.

Developmental exposures to methylmercury (MeHg) have life-long behavioral effects. Many micronutrients, including selenium, are involved in cellular defenses against oxidative stress and may reduce the severity of MeHg-induced deficits. Zebrafish embryos (<4 h post fertilization, hpf) were exposed to combinations of 0.0-0.30 microM MeHg and/or selenomethionine (SeMet) until 24 hpf then placed in clean medium. Fish were tested as adults under low light conditions ( approximately 60 microW/m(2)) for visual responses to a rotating black bar. Dose-dependent responses to MeHg exposure were evident (ANOVA, P<0.001) as evidenced by reduced responsiveness, whereas SeMet did not induce deficits except at 0.3 microM. Ratios of SeMet:MeHg of 1:1 or 1:3 resulted in responses that were indistinguishable from controls (ANOVA, P<0.001). No gross histopathologies were observed (H&E stain) in the retina or optic tectum at any MeHg concentration. Whole-cell, voltage-gated, depolarization-elicited outward K(+) currents of bipolar cells in intact retina of slices adult zebrafish were recorded and outward K(+) current amplitude was larger in bipolar cells of MeHg-treated fish. This was due to the intense response of cells expressing the delayed rectifying I(K) current; cells expressing the transient I(A) current displayed a slight trend for smaller amplitude among MeHg-treated fish. Developmental co-exposure to SeMet reduced but did not eliminate the increase in the MeHg-induced I(K) response, however, I(A) responses increased significantly over MeHg-treated fish to match control levels. Electrophysiological deficits parallel behavioral patterns in MeHg-treated fish, i.e., initial reactions to the rotating bar were followed by periods of inactivity and then a resumption of responses.

Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary: insights into TCDD-induced endocrine disruption and reproductive toxicity.

TCDD is a reproductive toxicant and endocrine disruptor, yet the mechanisms by which it causes these reproductive alterations are not fully understood. In order to provide additional insight into the molecular mechanisms that underlie TCDD's reproductive toxicity, we assessed TCDD-induced transcriptional changes in the ovary as they relate to previously described impacts on serum estradiol concentrations and altered follicular development in zebrafish. In silico computational approaches were used to correlate candidate regulatory motifs with observed changes in gene expression. Our data suggest that TCDD inhibits follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis, and that interference of estrogen-regulated signal transduction may also contribute to TCDD's impacts on follicular development. TCDD may also alter ovarian function by disrupting various signaling pathways such as glucose and lipid metabolism, and regulation of transcription. Furthermore, events downstream from initial TCDD molecular-targets likely contribute to ovarian toxicity following chronic exposure to TCDD. Data presented here provide further insight into the mechanisms by which TCDD disrupts follicular development and reproduction in fish, and can be used to formulate new hypotheses regarding previously documented ovarian toxicity.

Detection of mercury in aquatic environments using EPRE reporter zebrafish.

It has been proposed that transgenic zebrafish could be designed to detect low levels of chemical contaminants that cause oxidative stress in aquatic environments, such as heavy metals or pesticides. In this paper, we describe such a transgenic zebrafish that produces a luciferase-green fluorescent protein (LUC-GFP) fusion protein under conditions of oxidative stress. The reporter gene expression is under the regulation of the electrophile responsive element (EPRE), which activates gene expression in response to oxidative stressors. The GFP component of this fusion protein allows us to visually detect reporter gene activity in live animals to determine if activity is localized to a particular tissue. The luciferase component is capable of returning a quantitative assessment of reporter gene activity that allows us to determine if reporter gene activity is directly correlated to the concentration of the chemical inducer. We have tested this reporter construct in both transient and stable transgenic fish after exposure to a range of HgCl(2) concentrations. GFP expression from the EPRE-LUC-GFP construct was inducible in transient assays but was below the limit of detection in stable lines. In contrast, we observed inducible luciferase activity in both transient assays and stable lines treated with HgCl(2). We conclude that the EPRE is capable of driving reporter gene expression in a whole animal assay under conditions of oxidative stress. Furthermore, expression was induced at HgCl(2) concentrations that do not result in obvious morphological defects, making this approach useful for the detection of low levels of oxidative contaminants in aquatic environments.

Female reproductive impacts of dietary methylmercury in yellow perch (Perca flavescens) and zebrafish (Danio rerio).

The purpose of this study was to evaluate the effects of environmentally relevant dietary MeHg exposures on adult female yellow perch (Perca flavescens) and female zebrafish (Danio rerio) ovarian development and reproduction. Yellow perch were used in the study for their socioeconomic and ecological importance within the Great Lakes basin, and the use of zebrafish allowed for a detailed analysis of the molecular effects of MeHg following a whole life-cycle exposure. Chronic whole life dietary exposure of F1 zebrafish to MeHg mimics realistic wildlife exposure scenarios, and the twenty-week adult yellow perch exposure (where whole life-cycle exposures are difficult) captures early seasonal ovarian development. For both species, target dietary accumulation values were achieved prior to analyses. In zebrafish, several genes involved in reproductive processes were shown to be dysregulated by RNA-sequencing and quantitative real-time polymerase chain reaction (QPCR), but no significant phenotypic changes were observed regarding ovarian staging, fecundity, or embryo mortality. Yellow perch were exposed to dietary MeHg for 12, 16, or 20 weeks. In this species, a set of eight genes were assessed by QPCR in the pituitary, liver, and ovary, and no exposure-related changes were observed. The lack of genomic resources in yellow perch hinders the characterization of subtle molecular impacts. The ovarian somatic index, circulating estradiol and testosterone, and ovarian staging were not significantly altered by MeHg exposure in yellow perch. These results suggest that environmentally relevant MeHg exposures do not drastically reduce the reproductively important endpoints in these fish, but to capture realistic exposure scenarios, whole life-cycle yellow perch exposures are needed.

Developmental Methylmercury Exposure Affects Swimming Behavior and Foraging Efficiency of Yellow Perch (Perca flavescens) Larvae.

Methylmercury (MeHg) is a pervasive and ubiquitous environmental neurotoxicant within aquatic ecosystems, known to alter behavior in fish and other vertebrates. This study sought to assess the behavioral effects of developmental MeHg exposure on larval yellow perch (Perca flavescens)-a nonmodel fish species native to the Great Lakes. Embryos were exposed to MeHg (0, 30, 100, 300, and 1000 nM) for 20 h and then reared to 25 days post fertilization (dpf) for analyses of spontaneous swimming, visual motor response (VMR), and foraging efficiency. MeHg exposures rendered total mercury (THg) body burdens of 0.02, 0.21, 0.95, 3.14, and 14.93 µg/g (wet weight). Organisms exposed to 1000 nM exhibited high mortality; thus, they were excluded from downstream behavioral analyses. All MeHg exposures tested were associated with a reduction in spontaneous swimming at 17 and 25 dpf. Exposure to 30 and 100 nM MeHg caused altered locomotor output during the VMR assay at 21 dpf, whereas exposure to 100 nM MeHg was associated with decreased foraging efficiency at 25 dpf. For the sake of comparison, the second-lowest exposure tested here rendered a THg burden that represents the permissible level of consumable fish in the United States. Moreover, this dose is reported in roughly two-thirds of consumable fish species monitored in the United States, according to the Food and Drug Administration. Although the THg body burdens reported here were higher than expected in the environment, our study is the first to analyze the effects of MeHg exposure on fundamental survival behaviors of yellow perch larvae and advances in the exploration of the ecological relevance of behavioral end points.

Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

Gene expression changes related to endocrine function and decline in reproduction in fathead minnow (Pimephales promelas) after dietary methylmercury exposure.

BACKGROUND: Methylmercury (MeHg) is a known neurotoxic agent, but the mechanisms by which MeHg may act on reproductive pathways are relatively unknown. Several studies have indicated potential changes in hormone levels as well as declines in vertebrates with increasing dietary MeHg exposure. OBJECTIVES: The purpose of this study was to identify alterations in gene expression associated with MeHg exposure, specifically those associated with previously observed changes in reproduction and reproductive biomarkers. Fathead minnows, Pimephales promelas, were fed one of three diets that were similar to documented concentrations of MeHg in the diets of wild invertivorous and piscivorous fish. We used a commercial macroarray in conjunction with quantitative polymerase chain reaction to examine gene expression in fish in relation to exposure to these environmentally relevant doses of MeHg. RESULTS: Expression of genes commonly associated with endocrine disruption was altered with Hg exposure. Specifically, we observed a marked up-regulation in vitellogenin mRNA in individual Hg-exposed males and a significant decline in vitellogenin gene expression in female fish with increasing Hg concentrations. Other genes identified by the macroarray experiment included those associated with egg fertilization and development, sugar metabolism, apoptosis, and electron transport. We also observed differences in expression patterns between male and female fish not related to genes specifically associated with reproduction, indicating a potential physiological difference in the reaction of males and females to MeHg. CONCLUSION: Gene expression data may provide insight into the mechanisms by which MeHg affects reproduction in fish and indicate how MeHg differs in its effect from other heavy metals and endocrine-disrupting compounds.

Inhibition of follicular development, vitellogenesis, and serum 17beta-estradiol concentrations in zebrafish following chronic, sublethal dietary exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a potent endocrine disruptor with the ability to affect several biologic processes, including reproduction. In fish, sublethal exposure to TCDD is known to modulate overall reproductive capacity, but impacts on follicular development and vitellogenesis are unknown. Here we show that chronic, dietary exposure to 0.08, 0.32, or 0.80 ng TCDD female(-1) day(-1) decreased egg production by more than 50% and that spawning success was reduced by as much as 96%. Serum estradiol concentrations were decreased more than twofold, accounting, in part, for observed decreases in serum vitellogenin concentrations by as much as 29%. Our data suggest that decreased egg production is likely the result of TCDD-mediated inhibition of the transition from pre-vitellogenic stage follicles to vitellogenic stage follicles, as well as the induction of follicular atresia. The majority of reproductive toxicity of TCDD is likely due to direct impacts on the ovary, yet histopathologic observations suggest liver toxicity could also contribute to observed impacts on vitellogenesis. Importantly, even when overall egg production is not significantly affected, our data show that subtle physiologic changes induced by TCDD can lead to altered gonadogenesis. This suggests that long-term exposure to very low concentrations of TCDD could greatly affect fecundity and reproductive success in fishes.

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury.

This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30-240µM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1µM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03µM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.

Maternal methylmercury from a wild-caught walleye diet induces developmental abnormalities in zebrafish.

Maternal methylmercury (MeHg) exposure from a contaminated diet causes adverse effects in offspring, but the underlying mechanism(s) remains unclear. In the present study, we investigated the effects of maternal dietary MeHg-exposure on the offspring, using the zebrafish (Danio rerio) as a model system. Female zebrafish were exposed to MeHg (0.88-3.10ppm) by consuming a diet made from wild-caught walleye originally intended for human consumption. While dietary MeHg exposure did not significantly influence fecundity, offspring showed increases in morphologic alterations and mortality, neurobehavioral dysfunction, and dysregulation of global gene expression. Gene expression analysis suggested that MeHg might affect neuronal and muscular development via dysregulation of genes related to transcriptional regulation (such as supt5h) and cell cycle (such as ccnb1). Results from this study provide evidence that food intended for human consumption, with relatively modest levels of MeHg, may induce adverse effects in offspring.

Accumulation, tissue distribution, and maternal transfer of dietary 2,3,7,8,-tetrachlorodibenzo-p-dioxin: impacts on reproductive success of zebrafish.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is a reproductive toxicant and endocrine disruptor in nearly all vertebrates; however, the mechanisms by which TCDD alters the reproductive system is not well understood. The zebrafish provides a powerful vertebrate model system to investigate molecular mechanisms by which TCDD affects the reproductive system, but little is known regarding reproductive toxic response of zebrafish following chronic, sublethal exposure to TCDD. Here we investigate the accumulation of TCDD in selected tissues of adult female zebrafish and maternal transfer to offspring following dietary exposure to TCDD (0.08-2.16 ng TCDD/fish/day). TCDD accumulated in tissues of zebrafish in a dose- and time-dependent manner, except for brain. Chronic dietary exposure resulting in the accumulation of 1.1-36 ng/g fish did not induce an overt toxic response or suppress spawning activity. The ovosomatic index was impacted with an accumulation of as little as 0.6 ng/g fish, and 10% of the females showed signs of ovarian necrosis following accumulation of approximately 3 ng/g TCDD. Offspring health was impacted with an accumulation of as little as 1.1 ng/g female; thus the lowest observed effect level (LOEL) for reproductive toxicity in female zebrafish is approximately 0.6-1.1 ng/g fish. Maternal transfer resulted in the accumulation of 0.094-1.2 ng/g, TCDD, which was sufficient to induce the typical endpoints of larval TCDD toxicity, commonly referred to as blue sac syndrome. This study provides the necessary framework to utilize the zebrafish model system for further investigations into the molecular mechanisms by which TCDD exerts its reproductive toxic responses.