Developmental Pb exposure increases AD risk via altered intracellular Ca2+ homeostasis in hiPSC-derived cortical neurons.

Exposure to environmental chemicals such as lead (Pb) during vulnerable developmental periods can result in adverse health outcomes later in life. Human cohort studies have demonstrated associations between developmental Pb exposure and Alzheimer's disease (AD) onset in later life which were further corroborated by findings from animal studies. The molecular pathway linking developmental Pb exposure and increased AD risk, however, remains elusive. In this work, we used human iPSC-derived cortical neurons as a model system to study the effects of Pb exposure on AD-like pathogenesis in human cortical neurons. We exposed neural progenitor cells derived from human iPSC to 0, 15, and 50 ppb Pb for 48 h, removed Pb-containing medium, and further differentiated them into cortical neurons. Immunofluorescence, Western blotting, RNA-sequencing, ELISA, and FRET reporter cell lines were used to determine changes in AD-like pathogenesis in differentiated cortical neurons. Exposing neural progenitor cells to low-dose Pb, mimicking a developmental exposure, can result in altered neurite morphology. Differentiated neurons exhibit altered calcium homeostasis, synaptic plasticity, and epigenetic landscape along with elevated AD-like pathogenesis markers, including phosphorylated tau, tau aggregates, and Aß42/40. Collectively, our findings provide an evidence base for Ca dysregulation caused by developmental Pb exposure as a plausible molecular mechanism accounting for increased AD risk in populations with developmental Pb exposure.

Differential Developmental Neurotoxicity and Tissue Uptake of the Per- and Polyfluoroalkyl Substance Alternatives, GenX and PFBS.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic chemicals with several applications. Multiple adverse health effects are reported for longer carbon chain (≤C8) PFAS. Shorter carbon chain PFAS, [e.g., hexafluoropropylene oxide dimer acid (HFPO-DA; GenX) and perfluorobutanesulfonic acid (PFBS)] were introduced as alternatives. Past studies indicate that longer-chain PFAS are neurotoxic targeting the dopamine pathway, but it is not known if shorter-chain PFAS act similarly. This study aimed to evaluate developmental neurotoxicity and tissue uptake of GenX and PFBS using the zebrafish (Danio rerio). First, acute toxicity was assessed by measuring LC50 at 120 h postfertilization (hpf). Body burden was determined after embryonic exposure (1-72 hpf) to sublethal concentrations of GenX or PFBS by LC-ESI-MS/MS. Locomotor activity using a visual motor response assay at 120 hpf and dopamine levels at 72 hpf was assessed after embryonic exposure. PFBS was more acutely toxic and bioaccumulative than GenX. GenX and PFBS caused hyperactivity at 120 hpf, but stronger behavioral alterations were observed for PFBS. An increase in whole organism dopamine occurred at 40 ppb of GenX, while a decrease was observed at 400 ppb of PFBS. Differences detected in dopamine for these two PFAS indicate differential mechanisms of developmental neurotoxicity.

Impact of the hepatoselective glucokinase activator TTP399 on ketoacidosis during insulin withdrawal in people with type 1 diabetes.

AIMS: To determine the effect of TTP399, a hepatoselective glucokinase activator, on the risk of ketoacidosis during insulin withdrawal in individuals with type 1 diabetes (T1D). MATERIALS AND METHODS: Twenty-three participants with T1D using insulin pump therapy were randomized to 800 mg TTP399 (n = 12) or placebo (n = 11) for 7 to 10 days. After the treatment period, an insulin withdrawal test (IWT) was performed, during which insulin pumps were removed to induce ketogenesis. The IWT was stopped after 10 hours or if blood glucose reached >399 mg/dL [22.1 mmol/L], if beta-hydroxybutyrate (BHB) was >3.0 mmol/L, or for patient discomfort. The primary endpoint was the proportion of participants who reached BHB concentrations of 1 mmol/L or greater. RESULTS: During the 7- to 10-day treatment period, mean fasting plasma glucose was significantly reduced ( -27.6 vs. -4.4 mg/dL [-1.5 vs. -0.2 mmol/L]; P = 0.03) and there were fewer adverse events, including hypoglycaemia, in the TTP399-treated arm. During the IWT, no differences were observed between TTP399 and placebo in mean serum BHB concentration, mean duration of IWT, or BHB at termination of IWT. However, serum bicarbonate was numerically higher and urine acetoacetate was quantitatively lower in the TTP399-treated participants. As a result of higher bicarbonate values, none of the TTP399-treated participants met the prespecified criteria for diabetic ketoacidosis (DKA), defined as BHB >3 mmol/L and serum bicarbonate <18 mEq/L, compared to 42% of placebo-treated participants. CONCLUSIONS: When used as an adjunctive therapy to insulin, TTP399 improves glycaemia without increasing hypoglycaemia in individuals with T1D. During acute insulin withdrawal, TTP399 did not increase BHB concentrations and decreased the incidence of DKA.

Embryonic atrazine exposure and later in life behavioral and brain transcriptomic, epigenetic, and pathological alterations in adult male zebrafish.

Atrazine (ATZ), a commonly used pesticide linked to endocrine disruption, cancer, and altered neurochemistry, frequently contaminates water sources at levels above the US Environmental Protection Agency's 3 parts per billion (ppb; µg/L) maximum contaminant level. Adult male zebrafish behavior, brain transcriptome, brain methylation status, and neuropathology were examined to test the hypothesis that embryonic ATZ exposure causes delayed neurotoxicity, according to the developmental origins of health and disease paradigm. Zebrafish (Danio rerio) embryos were exposed to 0 ppb, 0.3 ppb, 3 ppb, or 30 ppb ATZ during embryogenesis (1-72 h post fertilization (hpf)), then rinsed and raised to maturity. At 9 months post fertilization (mpf), males had decreased locomotor parameters during a battery of behavioral tests. Transcriptomic analysis identified altered gene expression in organismal development, cancer, and nervous and reproductive system development and function pathways and networks. The brain was evaluated histopathologically for morphometric differences, and decreased numbers of cells were identified in raphe populations. Global methylation levels were evaluated at 12 mpf, and the body length, body weight, and brain weight were measured at 14 mpf to evaluate effects of ATZ on mature brain size. No significant difference in genome methylation or brain size was observed. The results demonstrate that developmental exposure to ATZ does affect neurodevelopment and neural function in adult male zebrafish and raises concern for possible health effects in humans due to ATZ's environmental presence and persistence. Graphical abstract.

Comparison of zebrafish in vitro and in vivo developmental toxicity assessments of perfluoroalkyl acids (PFAAs).

Perfluoroalkyl acids (PFAAs) are persistent environmental contaminants that are associated with various adverse health outcomes. Perfluorooctanoic acid (PFOA) is one of the most prominently detected PFAAs in the environment, which is now replaced with shorter chain carbon compounds including perfluorohexanoic acid (PFHxA) and perfluorobutyric acid (PFBA). The aim of this study was to compare the toxicity of four PFAAs as a function of chain length and head group (carboxylate versus sulfonate) with in vitro and in vivo zebrafish assessments, which were subsequently compared to other cell and aquatic models. Mortality rate increased with chain length (PFOA > PFHxA ≫ PFBA) in both whole embryo/larvae and embryonic cell models. The sulfonate group enhanced toxicity with perfluorobutane sulfonate (PFBS) showing higher toxicity than PFBA and PFHxA in both larvae and cells. Toxicity trends were similar among different aquatic models, but sensitivities varied. Discrepancies with other zebrafish studies were confirmed to be associated with a lack of neutralization of acidic pH of dosing solutions in these other investigations, demonstrating the need for rigor in reporting pH of exposure solutions in all experiments. The zebrafish embryonic cell line was also found to be similar to most other cell lines regardless of exposure length. Overall, results agree with findings in other cell lines and organisms where longer chain length and sulfonate group increase toxicity, except in investigations not neutralizing the exposure solutions for these acidic compounds.

Exposure to the Heavy-Metal Lead Induces DNA Copy Number Alterations in Zebrafish Cells.

DNA copy number variants are associated with the development of complex neurological diseases and disorders including autism spectrum disorder, schizophrenia, Alzheimer's disease, and Parkinson's disease. Exposure to multiple environmental chemicals including various heavy metals is suggested as a risk factor in these neurological diseases and disorders, but few studies have addressed if heavy-metal exposure can result in de novo DNA copy number changes as a genetic mechanism contributing to these disease outcomes. In this study to further investigate the relationship between heavy-metal exposure and de novo copy number alterations (CNAs), zebrafish fibroblast cells were exposed to the neurotoxicant lead (Pb). A crystal violet assay was first used to determine exposure concentrations with >80% cell confluency. Then a zebrafish-specific array comparative genomic hybridization platform was used to detect CNAs following a 72 h Pb exposure (0.24, 2.4, or 24 µM). The Pb exposure resulted in 72 CNA amplifications ranging in size from 5 to 329 kb. No deletions were detected. CNAs resulted in 15 CNA regions (CNARs), leaving 7 singlet CNAs. Two of the singlets were within high repeat genomic locations. The number of CNAs tended to increase in a concentration-dependent manner. Several CNARs encompassed genes previously reported to have altered expression with Pb exposure, suggesting a mechanistic link. In addition, almost all genes are associated within a molecular network with amyloid precursor protein, a key molecular target associated with the pathophysiology of Alzheimer's disease. Overall, these findings show that Pb exposure results in de novo CNAs that could serve as a mechanism driving adverse health outcomes associated with Pb toxicity including neurological disease pathogenesis for further study.

Use of Zebrafish in Drug Discovery Toxicology.

Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.

Chemical and Genetic Zebrafish Models to Define Mechanisms of and Treatments for Dopaminergic Neurodegeneration.

The zebrafish (Danio rerio) is routinely used in biological studies as a vertebrate model system that provides unique strengths allowing applications in studies of neurodevelopmental and neurodegenerative diseases. One specific advantage is that the neurotransmitter systems are highly conserved throughout vertebrate evolution, including between zebrafish and humans. Disruption of the dopaminergic signaling pathway is linked to multiple neurological disorders. One of the most common is Parkinson's disease, a neurodegenerative disease associated with the loss of dopaminergic neurons, among other neuropathological characteristics. In this review, the development of the zebrafish's dopaminergic system, focusing on genetic control of the dopaminergic system, is detailed. Second, neurotoxicant models used to study dopaminergic neuronal loss, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the pesticides paraquat and rotenone, and 6-hydroxydopamine (6-OHDA), are described. Next, zebrafish genetic knockdown models of dj1, pink1, and prkn established for investigating mechanisms of Parkinson's disease are discussed. Chemical modulators of the dopaminergic system are also highlighted to showcase the applicability of the zebrafish to identify mechanisms and treatments for neurodegenerative diseases such as Parkinson's disease associated with the dopaminergic system.

Sex-specific characterization and evaluation of the Alzheimer's disease genetic risk factor sorl1 in zebrafish during aging and in the adult brain following a 100 ppb embryonic lead exposure.

Developmental lead (Pb) exposure is suggested in laboratory studies to be a trigger for neurodegenerative diseases such as Alzheimer's disease (AD). Sortilin-related receptor, L (DLR class) A repeats-containing (SORL1) is a recently identified AD genetic risk factor. SORL1 has limited characterization in vertebrate models in comparison to other AD genetic risk factors. To characterize SORL1 further, protein sequence homology between humans, mice and zebrafish was analyzed and showed conservation of functional repeats and domain orientation. Next, spatial expression of sorl1 in zebrafish larvae was completed and diffuse expression in neural tissue that was not restricted to the brain was observed. Influences of sex and age on quantitative expression of sorl1 in the brain of adult zebrafish were then assessed. Sex-specific alteration of sorl1 expression transpired during the aging process in females. The zebrafish was then utilized to investigate the impacts of a 100 ppb embryonic Pb exposure on sorl1 expression and other known AD genetic risk factors. Sex-specific quantitative gene expression analysis was completed with adult zebrafish brain to compare those developmentally exposed to Pb or a control treatment, but no significant difference in sorl1 expression or other AD genetic risk factors was observed. Overall, this study provided characterization of sorl1 with changes in brain expression during aging being female-specific. This finding is in agreement with females being more prone to the onset of AD, but analysis of additional AD genetic risk factors is needed to facilitate our understanding of the impact of a 100 ppb embryonic Pb exposure. Copyright © 2016 John Wiley & Sons, Ltd.

Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene.

Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint, more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models, with the majority of studies using relatively higher parts per million (mg/L) doses. In this study, to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; µg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin, and mitochondrial function were assessed. A significant dose-response decrease in angiogenesis, F-actin, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study support TCE as a developmental cardiovascular toxicant, provide molecular targets and pathways for investigation in future studies, and indicate a need for continued priority for environmental regulation.

Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model.

The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed.

Directional and color preference in adult zebrafish: Implications in behavioral and learning assays in neurotoxicology studies.

The zebrafish (Danio rerio) is a useful vertebrate model organism for neurological studies. While a number of behavior and learning assays are recently reported in the literature for zebrafish, many of these assays are still being refined. The initial purpose of this study was to apply a published T-maze assay for adult zebrafish that measures how quickly an organism can discriminate between different color stimuli after receiving reinforcement to measure learning in a study investigating the later life impacts of developmental Pb exposure. The original results were inconclusive as the control group showed a directional and color preference. To assess directional preference further, a three-chambered testing apparatus was constructed and rotated in several directions. The directional preference observed in males was alleviated by rotating the arms pointing west and east. In addition, color preference was investigated using all combinations of five different colors (orange, yellow, green, blue and purple). With directional preference alleviated results showed that both male and female zebrafish preferred colors of shorter wavelengths. An additional experiment tested changes in color preference due to developmental exposure to Pb in adult male zebrafish. Results revealed that Pb-exposed males gained and lost certain color preferences compared to control males and the preference for short wavelengths was decreased. Overall, these results show that consideration and pretesting should be completed before applying behavioral and learning assays involving adult zebrafish to avoid innate preferences and confounding changes in neurotoxicology studies and that developmental Pb exposure alters color preferences in adult male zebrafish.

Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis.

Copy number variants (CNVs) represent a substantial source of genomic variation in vertebrates and have been associated with numerous human diseases. Despite this, the extent of CNVs in the zebrafish, an important model for human disease, remains unknown. Using 80 zebrafish genomes, representing three commonly used laboratory strains and one native population, we constructed a genome-wide, high-resolution CNV map for the zebrafish comprising 6,080 CNV elements and encompassing 14.6% of the zebrafish reference genome. This amount of copy number variation is four times that previously observed in other vertebrates, including humans. Moreover, 69% of the CNV elements exhibited strain specificity, with the highest number observed for Tubingen. This variation likely arose, in part, from Tubingen's large founding size and composite population origin. Additional population genetic studies also provided important insight into the origins and substructure of these commonly used laboratory strains. This extensive variation among and within zebrafish strains may have functional effects that impact phenotype and, if not properly addressed, such extensive levels of germ-line variation and population substructure in this commonly used model organism can potentially confound studies intended for translation to human diseases.

Cerebral Vascular Toxicity after Developmental Exposure to Arsenic (As) and Lead (Pb) Mixtures.

Arsenic (As) and lead (Pb) are environmental pollutants found in common sites linked to similar adverse health effects. This study determined driving factors of neurotoxicity on the developing cerebral vasculature with As and Pb mixture exposures. Cerebral vascular toxicity was evaluated at mixture concentrations of As and Pb representing human exposures levels (10 or 100 parts per billion; ppb; µg/L) in developing zebrafish by assessing behavior, morphology, and gene expression. In the visual motor response assay, hyperactivity was observed in all three outcomes in dark phases in larvae with exposure (1-120 h post fertilization, hpf) to 10 ppb As, 10 ppb Pb, or 10 ppb mix treatment. Time spent moving exhibited hyperactivity in dark phases for 100 ppb As and 100 ppb mix treatment groups only. A decreased brain length and ratio of brain length to total length in the 10 ppb mix group was measured with no alterations in other treatment groups or other endpoints (i.e., total larval length, head length, or head width). Alternatively, measurements of cerebral vasculature in the midbrain and cerebellum uncovered decreased total vascularization at 72 hpf in all treatment groups in the mesencephalon and in all treatment groups, except the 100 ppb Pb and 10 ppb As groups, in the cerebellum. In addition, decreased sprouting and branching occurred in the mesencephalon, while only decreased branching was measured in the cerebellum. The 10 ppb Pb group showed several cerebral vasculature modifications that were aligned with a specific gene expression alteration pattern different from other treatment groups. Additionally, the 100 ppb As group drove gene alterations, along with several other endpoints, for changes observed in the 100 ppb mix treatment group. Perturbations assessed in this study displayed non-linear concentration-responses, which are important to consider in environmental health outcomes for As and Pb neurotoxicity.

Developmental neurotoxicity of PFOA exposure on hiPSC-derived cortical neurons.

PFOA is a legacy Per- and Polyfluorinated Substances (PFAS), a group of chemicals widely used in various industrial applications and consumer products. Although there has been a voluntary phase out of PFOA since 2005, it is still widely detected in various water supplies. A growing body of evidence suggests an association between PFOA exposure, particularly during developmental stages, with increased risks of neurodegenerative diseases (NDs). The neurotoxic mechanism of developmental PFOA exposure, however, remains poorly understood. Utilizing human induced-pluripotent stem cell (hiPSC)-derived cortical neurons, we investigated the effect of PFOA exposure prior to differentiation and assessed changes in neuronal characteristics, transcriptome, and neurodegeneration markers mimicking a Developmental Origin of Health and Disease (DoHAD) paradigm. Exposure to PFOA before neuron differentiation resulted in persistent alterations in nuclear morphology, neuronal network, and calcium activity. RNA sequencing analysis further revealed transcriptomic changes aligning with Alzheimer's Disease (AD) after PFOA exposure. These observations were further corroborated by alterations in tau phosphorylation markers, the presence of fibrillar tau, an increase in liquid droplets, and a decrease in RNA translational efficiency characterized using a battery of biochemical assays. Taken together, our results revealed persistent deficits of key neuronal characteristics induced by pre-differentiation PFOA exposure, suggesting impairments in several AD-related pathways that can together contribute to the elevation of AD risk after pre-differentiation PFOA exposure.

Elevated parkinsonism pathological markers in dopaminergic neurons with developmental exposure to atrazine.

Atrazine (ATZ) is one of the most used herbicides in the US and a known endocrine disruptor. ATZ is frequently detected in drinking water, especially in Midwestern regions of the United States, exceeding the EPA regulation of maximum contamination level (MCL) of 3 ppb. Epidemiology studies have suggested an association between ATZ exposure and neurodegeneration. Less, however, is known about the neurotoxic mechanism of ATZ, particularly for exposures at a developmental stage. Here, we exposed floor plate progenitors (FPPs) derived from human induced pluripotent stem cells (hiPSCs) to low concentrations of ATZ at 0.3 and 3 ppb for two days followed by differentiation into dopaminergic (DA) neurons in ATZ-free medium. We then examined the morphology, activity, pathological protein aggregation, and transcriptomic changes of differentiated DA neurons. We observed significant decrease in the complexity of neurite network, increase of neuronal activity, and elevated tau- and α-synuclein (aSyn) pathologies after ATZ exposure. The ATZ-induced neuronal changes observed here align with pathological characteristics in Parkinson's disease (PD). Transcriptomic analysis further corroborates our findings; and collectively provides a strong evidence base that low-concentration ATZ exposure during development can elicit increased risk of neurodegeneration.

Pre-differentiation GenX exposure induced neurotoxicity in human dopaminergic-like neurons.

GenX, also known as hexafluoropropylene oxide dimer acid (HFPO-DA) was introduced as a safer alternative to perfluorooctanoic acid (PFOA) in 2009. After nearly two decades of applications there are increasing safety concerns about GenX due to its association with various organ damages. Few studies, however, have systematically assessed the molecular neurotoxicity of low-dose GenX exposure. Here, we evaluated the effects of pre-differentiation exposure of GenX on dopaminergic (DA) -like neurons using SH-SY5Y cell line; and assessed changes in epigenome, mitochondrion, and neuronal characteristics. Low dose GenX exposure at 0.4 and 4 µg/L prior to differentiation induced persistent changes in nuclear morphology and chromatin arrangements, manifested specifically in the facultative repressive marker H3K27me3. We also observed impaired neuronal network, increased calcium activity along with alterations in Tyrosine hydroxylase (TH) and α-Synuclein (αSyn) after prior exposure to GenX. Collectively, our results identified neurotoxicity of low-dose GenX exposure in human DA-like neurons following a developmental exposure scheme. The observed changes in neuronal characteristics suggest GenX as a potential neurotoxin and risk factor for Parkinson's disease.

Adverse developmental impacts in progeny of zebrafish exposed to the agricultural herbicide atrazine during embryogenesis.

Atrazine (ATZ) is an herbicide commonly used on crops in the Midwestern US and other select global regions. The US Environmental Protection Agency ATZ regulatory limit is 3 parts per billion (ppb; µg/L), but this limit is often exceeded. ATZ has a long half-life, is a common contaminant of drinking water sources, and is indicated as an endocrine disrupting chemical in multiple species. The zebrafish was used to test the hypothesis that an embryonic parental ATZ exposure alters protein levels leading to modifications in morphology and behavior in developing progeny. Zebrafish embryos (F1) were collected from adults (F0) exposed to 0, 0.3, 3, or 30 ppb ATZ during embryogenesis. Differential proteomics, morphology, and behavior assays were completed with offspring aged 120 or 144 h with no additional chemical treatment. Proteomic analysis identified differential expression of proteins associated with neurological development and disease; and organ and organismal morphology, development, and injury, specifically the skeletomuscular system. Head length and ratio of head length to total length was significantly increased in the F1 of 0.3 and 30 ppb ATZ groups (p < 0.05). Based on molecular pathway alterations, further craniofacial morphology assessment found decreased distance for cartilaginous structures, decreased surface area and distance between saccular otoliths, and a more posteriorly positioned notochord (p < 0.05), indicating delayed ossification and skeletal growth. The visual motor response assay showed hyperactivity in progeny of the 30 ppb treatment group for distance moved and of the 0.3 and 30 ppb treatment groups for time spent moving (p < 0.05). Due to the changes in saccular otoliths, an acoustic startle assay was completed and showed decreased response in the 0.3 and 30 ppb treatments (p < 0.05). These findings suggest that a single embryonic parental exposure alters cellular pathways in their progeny that lead to perturbations in craniofacial development and behavior.

Global variation in copy number in the human genome.

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

Joint Action Toxicity of Arsenic (As) and Lead (Pb) Mixtures in Developing Zebrafish.

Arsenic (As) and lead (Pb) are environmental pollutants found in common sites and linked to similar adverse health effects. Multiple studies have investigated the toxicity of each metal individually or in complex mixtures. Studies defining the joint interaction of a binary exposure to As and Pb, especially during the earliest stages of development, are limited and lack confirmation of the predicted mixture interaction. We hypothesized that a mixture of As (iAsIII) and Pb will have a concentration addition (CA) interaction informed by common pathways of toxicity of the two metals. To test this hypothesis, developing zebrafish (1-120 h post fertilization; hpf) were first exposed to a wide range of concentrations of As or Pb separately to determine 120 hpf lethal concentrations. These data were then used in the CA and independent action (IA) models to predict the type of mixture interaction from a co-exposure to As and Pb. Three titration mixture experiments were completed to test prediction of observed As and Pb mixture interaction by keeping the Pb concentration constant and varying As concentrations in each experiment. The prediction accuracy of the two models was then calculated using the prediction deviation ratio (PDR) and Chi-square test and regression modeling applied to determine type of interaction. Individual metal exposures determined As and Pb concentrations at which 25% (39.0 ppm Pb, 40.2 ppm As), 50% (73.8 ppm Pb, 55.4 ppm As), 75% (99.9 ppm Pb, 66.6 ppm As), and 100% (121.7 ppm Pb, 77.3 ppm As) lethality was observed at 120 hpf. These data were used to graph the predicted mixture interaction using the CA and IA models. The titration experiments provided experimental observational data to assess the prediction. PDR values showed the CA model approached 1, whereas all PDR values for the IA model had large deviations from predicted data. In addition, the Chi-square test showed most observed results were significantly different from the predictions, except in the first experiment (Pb LC25 held constant) with the CA model. Regression modeling for the IA model showed primarily a synergistic response among all exposure scenarios, whereas the CA model indicated additive response at lower exposure concentrations and synergism at higher exposure concentrations. The CA model was a better predictor of the Pb and As binary mixture interaction compared to the IA model and was able to delineate types of mixture interactions among different binary exposure scenarios.