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.

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.

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.

Telomere dysfunction and DNA-PKcs deficiency: characterization and consequence.

The mechanisms by which cells accurately distinguish between DNA double-strand break (DSB) ends and telomeric DNA ends remain poorly defined. Recent investigations have revealed intriguing interactions between DNA repair and telomeres. We were the first to report a requirement for the nonhomologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PK) in the effective end-capping of mammalian telomeres. Here, we report our continued characterization of uncapped (as opposed to shortened) dysfunctional telomeres in cells deficient for the catalytic subunit of DNA-PK (DNA-PKcs) and shed light on their consequence. We present evidence in support of our model that uncapped telomeres in this repair-deficient background are inappropriately detected and processed as DSBs and thus participate not only in spontaneous telomere-telomere fusion but, importantly, also in ionizing radiation-induced telomere-DSB fusion events. We show that phosphorylation of DNA-PKcs itself (Thr-2609 cluster) is a critical event for proper telomere end-processing and that ligase IV (NHEJ) is required for uncapped telomere fusion. We also find uncapped telomeres in cells from the BALB/c mouse, which harbors two single-nucleotide polymorphisms that result in reduced DNA-PKcs abundance and activity, most markedly in mammary tissue, and are both radiosensitive and susceptible to radiogenic mammary cancer. Our results suggest mechanistic links between uncapped/dysfunctional telomeres in DNA-PKcs-deficient backgrounds, radiation-induced instability, and breast cancer. These studies provide the first direct evidence of genetic susceptibility and environmental insult interactions leading to a unique and ongoing form of genomic instability capable of driving carcinogenesis.