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.

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.

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.

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.

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.

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.

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.

Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains.

Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this "RAD-sex" method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, "male genotypes" became males and some "female genotypes" also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture.

Histopathologic alterations associated with global gene expression due to chronic dietary TCDD exposure in juvenile zebrafish.

The goal of this project was to investigate the effects and possible developmental disease implication of chronic dietary TCDD exposure on global gene expression anchored to histopathologic analysis in juvenile zebrafish by functional genomic, histopathologic and analytic chemistry methods. Specifically, juvenile zebrafish were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb, and fish were sampled following 0, 7, 14, 28 and 42 d after initiation of the exposure. TCDD accumulated in a dose- and time-dependent manner and 100 ppb TCDD caused TCDD accumulation in female (15.49 ppb) and male (18.04 ppb) fish at 28 d post exposure. Dietary TCDD caused multiple lesions in liver, kidney, intestine and ovary of zebrafish and functional dysregulation such as depletion of glycogen in liver, retrobulbar edema, degeneration of nasal neurosensory epithelium, underdevelopment of intestine, and diminution in the fraction of ovarian follicles containing vitellogenic oocytes. Importantly, lesions in nasal epithelium and evidence of endocrine disruption based on alternatively spliced vasa transcripts are two novel and significant results of this study. Microarray gene expression analysis comparing vehicle control to dietary TCDD revealed dysregulated genes involved in pathways associated with cardiac necrosis/cell death, cardiac fibrosis, renal necrosis/cell death and liver necrosis/cell death. These baseline toxicological effects provide evidence for the potential mechanisms of developmental dysfunctions induced by TCDD and vasa as a biomarker for ovarian developmental disruption.

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.

Low-dose gold nanoparticles exert subtle endocrine-modulating effects on the ovarian steroidogenic pathway ex vivo independent of oxidative stress.

Gold nanoparticles (GNPs) have gained considerable attention for application in science and industry. However, the untoward effects of such particles on female fertility remain unclear. The objectives of this study were to (1) examine the effects of 10-nm GNPs on progesterone and estradiol-17ß accumulation by rat ovaries ex vivo and (2) to identify the locus/loci whereby GNPs modulate steroidogenesis via multiple-reference gene quantitative real-time RT-PCR. Regression analyses indicated a positive relationship between both Star (p < 0.05, r(2) = 0.278) and Cyp11a1 (p < 0.001, r(2) = 0.366) expression and P4 accumulation upon exposure to 1.43 × 10(6) GNPs/mL. Additional analyses showed that E2 accumulation was positively associated with Hsd3b1 (p < 0.05, r(2) = 0.181) and Cyp17a1 (p < 0.01, r(2) = 0.301) expression upon exposure to 1.43 × 1(3) and 1.43 × 10(9) GNPs/mL, respectively. These results suggest a subtle treatment-dependent impact of low-dose GNPs on the relationship between progesterone or estradiol-17ß and specific steroidogenic target genes, independent of oxidative stress or inhibin.

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

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.

Effects of methylmercury on epigenetic markers in three model species: mink, chicken and yellow perch.

We previously reported that methylmercury (MeHg) exposure is associated with DNA hypomethylation in the brain stem of male polar bears. Here, we conveniently use archived tissues obtained from controlled laboratory exposure studies to look for evidence that MeHg can disrupt DNA methylation across taxa. Brain (cerebrum) tissues from MeHg-exposed mink (Neovison vison), chicken (Gallus gallus) and yellow perch (Perca flavescens) were analyzed for total Hg levels and global DNA methylation. Tissues from chicken and mink, but not perch, were also analyzed for DNA methyltransferase (DNMT) activity. In mink we observed significant reductions in global DNA methylation in an environmentally-relevant dietary exposure group (1 ppm MeHg), but not in a higher group (2 ppm MeHg). DNMT activity was significantly reduced in all treatment groups. In chicken or yellow perch, no statistically significant effects of MeHg were observed. Dose-dependent trends were observed in the chicken data but the direction of the change was not consistent between the two endpoints. Our results suggest that MeHg can be epigenetically active in that it has the capacity to affect DNA methylation in mammals. The variability in results across species may suggest inter-taxa differences in epigenetic responses to MeHg, or may be related to differences among the exposure scenarios used as animals were exposed to MeHg through different routes (dietary, egg injection), for different periods of time (19-89 days) and at different life stages (embryonic, juvenile, adult).

Comparison of neurobehavioral effects of methylmercury exposure in older and younger adult zebrafish (Danio rerio).

It is widely recognized that the nature and severity of responses to toxic exposure are age-dependent. Using active avoidance conditioning as the behavioral paradigm, the present study examined the effect of short-term methylmercury (MeHg) exposure on two adult age classes, 1- and 2-year-olds to coincide with zebrafish in relatively peak vs. declining health conditions. In Experiment 1, 2-year-old zebrafish were randomly divided into groups and were exposed to no MeHg, 0.15% ethanol (EtOH), 0.01, 0.03, 0.1, or 0.3 µM of MeHg (in 0.15% ethanol) for 2 weeks. The groups were then trained and tested for avoidance responses. The results showed that older zebrafish exposed to no MeHg or EtOH learned and retained avoidance responses. However, 0.01 µM or higher concentrations of MeHg exposure impaired avoidance learning in a dose-dependent manner with 0.3 µM of MeHg exposure producing death during the exposure period or shortly after the exposure but before the avoidance training. In Experiment 2, 1-year-old zebrafish were randomly divided into groups and were exposed to the same concentrations of MeHg used in Experiment 1 for 2 weeks. The groups were then trained and tested for avoidance responses. The results showed that younger zebrafish exposed to no MeHg, EtOH, or 0.01 µM of MeHg learned and retained avoidance responses, while 0.1 or 0.3 µM of MeHg exposure impaired avoidance learning in a dose-dependent manner. The study suggested that MeHg exposure produced learning impairments at a much lower concentration of MeHg exposure and more severely in older adult compared against younger adult zebrafish even after short exposure times.