John Cowden

Gene expression changes in developing zebrafish as potential markers for rapid developmental neurotoxicity screening

Hazard information essential to guide developmental neurotoxicity risk assessments is limited for many chemicals. As developmental neurotoxicity testing using rodents is laborious and expensive, alternative species such as zebrafish are being adapted for rapid toxicity screening. Assessing the developmental neurotoxicity potential of chemicals in a rapid throughput mode will be aided by the identification and characterization of transcriptional biomarkers that can be measured accurately and rapidly. To this end, the developmental expression profiles of ten nervous system genes were characterized in 1 to 6 days post fertilization zebrafish embryos/larvae using quantitative real time PCR (qRT-PCR). Transcripts of synapsinII a (syn2a) and myelin basic protein (mbp) increased throughout development, while transcripts of gap43, elavl3, nkx2.2a, neurogenin1 (ngn1), alpha1-tubulin, and glial fibrillary acidic protein (gfap) initially increased, but subsequently declined. Transcripts for nestin and sonic hedgehog a (shha) decreased during development. We tested the responses of these potential biomarkers to developmental neurotoxicant exposure, and found that the expression profiles of a subset of genes were altered both during and after exposure to sublethal doses of ethanol, a known developmental neurotoxicant. Collectively, these data indicate that transcript levels of the candidate genes change during development in patterns which are consistent with literature reports, and that the expression of the transcripts is perturbed by treatment with a developmental neurotoxicant (ethanol). These results suggest that the expression profiles of these genes may be useful biomarkers for rapid evaluation of the developmental neurotoxicity potential of chemicals.

Arsenic and Environmental Health: State of the Science and Future Research Opportunities

Background: Exposure to inorganic and organic arsenic compounds is a major public health problem that affects hundreds of millions of people worldwide. Exposure to arsenic is associated with cancer and noncancer effects in nearly every organ in the body, and evidence is mounting for health effects at lower levels of arsenic exposure than previously thought. Building from a tremendous knowledge base with > 1,000 scientific papers published annually with “arsenic” in the title, the question becomes, what questions would best drive future research directions? Objectives: The objective is to discuss emerging issues in arsenic research and identify data gaps across disciplines. Methods: The National Institutes of Health’s National Institute of Environmental Health Sciences Superfund Research Program convened a workshop to identify emerging issues and research needs to address the multi-faceted challenges related to arsenic and environmental health. This review summarizes information captured during the workshop. Discussion: More information about aggregate exposure to arsenic is needed, including the amount and forms of arsenic found in foods. New strategies for mitigating arsenic exposures and related health effects range from engineered filtering systems to phytogenetics and nutritional interventions. Furthermore, integration of omics data with mechanistic and epidemiological data is a key step toward the goal of linking biomarkers of exposure and susceptibility to disease mechanisms and outcomes. Conclusions: Promising research strategies and technologies for arsenic exposure and adverse health effect mitigation are being pursued, and future research is moving toward deeper collaborations and integration of information across disciplines to address data gaps. Citation: Carlin DJ, Naujokas MF, Bradham KD, Cowden J, Heacock M, Henry HF, Lee JS, Thomas DJ, Thompson C, Tokar EJ, Waalkes MP, Birnbaum LS, Suk WA. 2016. Arsenic and environmental health: state of the science and future research opportunities. Environ Health Perspect 124:890–899; http://dx.doi.org/10.1289/ehp.1510209

AS3MT, GSTO, and PNP polymorphisms: impact on arsenic methylation and implications for disease susceptibility.

BACKGROUND Oral exposure to inorganic arsenic (iAs) is associated with adverse health effects. Epidemiological studies suggest differences in susceptibility to these health effects, possibly due to genotypic variation. Genetic polymorphisms in iAs metabolism could lead to increased susceptibility by altering urinary iAs metabolite concentrations. OBJECTIVE To examine the impact of genotypic polymorphisms on iAs metabolism. METHODS We screened 360 publications from PubMed and Web of Science for data on urinary mono- and dimethylated arsenic (MMA and DMA) percentages and polymorphic genes encoding proteins that are hypothesized to play roles in arsenic metabolism. The genes we examined were arsenic (+3) methyltransferase (AS3MT), glutathione-s-transferase omega (GSTO), and purine nucleoside phosphorylase (PNP). Relevant data were pooled to determine which polymorphisms are associated across studies with changes in urinary metabolite concentration. RESULTS In our review, AS3MT polymorphisms rs3740390, rs11191439, and rs11191453 were associated with statistically significant changes in percent urinary MMA. Studies of GSTO polymorphisms did not indicate statistically significant associations with methylation, and there are insufficient data on PNP polymorphisms to evaluate their impact on metabolism. DISCUSSION Collectively, these data support the hypothesis that AS3MT polymorphisms alter in vivo metabolite concentrations. Preliminary evidence suggests that AS3MT genetic polymorphisms may impact disease susceptibility. GSTO polymorphisms were not associated with iAs-associated health outcomes. Additional data are needed to evaluate the association between PNP polymorphisms and iAs-associated health outcomes. Delineation of these relationships may inform iAs mode(s) of action and the approach for evaluating low-dose health effects for iAs. CONCLUSIONS Genotype impacts urinary iAs metabolite concentrations and may be a potential mechanism for iAs-related disease susceptibility.

Developmental exposure to valproate and ethanol alters locomotor activity and retino-tectal projection area in zebrafish embryos.

Given the minimal developmental neurotoxicity data available for the large number of new and existing chemicals, there is a critical need for alternative methods to identify and prioritize chemicals for further testing. We outline a developmental neurotoxicity screening approach using zebrafish embryos. Embryos were exposed to nominal concentrations of either valproate or ethanol then examined for lethality, malformation, nervous system structure and locomotor activity. Developmental valproate exposure caused locomotor activity changes at concentrations that did not result in malformations and showed a concentration-dependent decrease in retino-tectal projection area in the optic tectum. Developmental ethanol exposure also affected retino-tectal projection area at concentrations below those concentrations causing malformations. As both valproate and ethanol are known human developmental neurotoxicants, these results add to the growing body of evidence showing the potential utility of zebrafish in screening compounds for mammalian developmental neurotoxicity.

Developmental Neurotoxicity of Engineered Nanomaterials: Identifying Research Needs to Support Human Health Risk Assessment

Increasing use of engineered nanomaterials (ENM) in consumer products and commercial applications has helped drive a rise in research related to the environmental health and safety (EHS) of these materials. Within the cacophony of information on ENM EHS to date are data indicating that these materials may be neurotoxic in adult animals. Evidence of elevated inflammatory responses, increased oxidative stress levels, alterations in neuronal function, and changes in cell morphology in adult animals suggests that ENM exposure during development could elicit developmental neurotoxicity (DNT), especially considering the greater vulnerability of the developing brain to some toxic insults. In this review, we examine current findings related to developmental neurotoxic effects of ENM in the context of identifying research gaps for future risk assessments. The basic risk assessment paradigm is presented, with an emphasis on problem formulation and assessments of exposure, hazard, and dose response for DNT. Limited evidence suggests that in utero and postpartum exposures are possible, while fewer than 10 animal studies have evaluated DNT, with results indicating changes in synaptic plasticity, gene expression, and neurobehavior. Based on the available information, we use current testing guidelines to highlight research gaps that may inform ENM research efforts to develop data for higher throughput methods and future risk assessments for DNT. Although the available evidence is not strong enough to reach conclusions about DNT risk from ENM exposure, the data indicate that consideration of ENM developmental neurotoxic potential is warranted.

Use of Medaka in Toxicity Testing

Small aquarium fishes are increasingly used as animal models, and one of these, the Japanese Medaka (Oryzias latipes), is frequently utilized for toxicity testing. While these vertebrates have many similarities with their terrestrial counterparts, there are differences that must be considered if these organisms are to be used to their highest potential. Commonly, testing may employ either the developing embryo or adults; both are easy to use and work with. To illustrate the utility and breadth of toxicity testing possible using medaka fish, we present protocols for assessing neurotoxicity in developing embryos, evaluating toxicant effects on sexual phenotype after treatment with endocrine‐disrupting chemicals by sexual genotyping, and measuring hepatotoxicity in adult fish after treatment with a model hepatotoxicant. The methods run the gamut from immunohistology through PCR to basic histological techniques. Curr. Protoc. Toxicol. 39:1.10.1‐1.10.36.

Relationships between arsenic concentrations in drinking water and lung and bladder cancer incidence in U.S. counties

Increased risks of lung and bladder cancer have been observed in populations exposed to high levels of inorganic arsenic. However, studies at lower exposures (i.e., less than 100 μg/l in water) have shown inconsistent results. We therefore conducted an ecological analysis of the association between historical drinking water arsenic concentrations and lung and bladder cancer incidence in U.S. counties. We used drinking water arsenic concentrations measured by the U.S. Geological Survey and state agencies in the 1980s and 1990s as proxies for historical exposures in counties where public groundwater systems and private wells are important sources of drinking water. Relationships between arsenic levels and cancer incidence in 2006–2010 were explored by Poisson regression analyses, adjusted for groundwater dependence and important demographic covariates. The median and 95th percentile county mean arsenic concentrations were 1.5 and 15.4 μg/l, respectively. Water arsenic concentrations were significant and positively associated with female and male bladder cancer, and with female lung cancer. Our findings support an association between low water arsenic concentrations and lung and bladder cancer incidence in the United States. However, the limitations of the ecological study design suggest caution in interpreting these results.

Systematic review of differential inorganic arsenic exposure in minority, low-income, and indigenous populations in the United States

Inorganic arsenic (iAs) is a human carcinogen and associated with cardiovascular, respiratory, and skin diseases. Natural and anthropogenic sources contribute to low concentrations of iAs in water, food, soil, and air. Differential exposure to environmental hazards in minority, indigenous, and low income populations is considered an environmental justice (EJ) concern, yet it is unclear if higher iAs exposure occurs in these populations. A systematic review was conducted to evaluate evidence for differential iAs exposure in the United States (US). The peer-reviewed literature was searched for studies that (1) estimated iAs exposure based on environmental concentrations of iAs in water, food, soil, or iAs biomarkers and (2) examined iAs exposure in minority, indigenous, and low income US populations. Five studies were identified that estimated exposures and provided demographic information about EJ populations. These studies reported arsenic concentrations in water, soil, or food to estimate exposure, with varied evidence of differential exposure. Additionally, six studies were identified that suggested potential arsenic exposure from environmental sources including soil, rice, private well-water, and fish, but did not report data stratified by demographic information. Evidence across these 11 studies was qualitatively integrated to draw conclusions about differential iAs exposure. The total body of evidence is limited by lack of individual exposure measures, lack of iAs concentration data, and insufficient comparative demographic data. Based upon these data gaps, there is inadequate evidence to conclude whether differential exposure to iAs is an EJ concern in the US.

Generation and characterization of neurogenin1-GFP transgenic medaka with potential for rapid developmental neurotoxicity screening.

Fish models such as zebrafish and medaka are increasingly used as alternatives to rodents in developmental and toxicological studies. These developmental and toxicological studies can be facilitated by the use of transgenic reporters that permit the real-time, noninvasive observation of the fish. Here we report the construction and characterization of transgenic medaka lines expressing green fluorescent protein (GFP) under the control of the zebrafish neurogenin 1 (ngn1) gene promoter. Neurogenin (ngn1) is a helix-loop-helix transcription factor expressed in proliferating neuronal progenitor cells early in neuronal differentiation and plays a crucial role in directing neurogenesis. GFP expression was detected from 24 h post-fertilization until hatching, in a spatial pattern consistent with the previously reported zebrafish ngn1 expression. Temporal expression of the transgene parallels the expression profile of the endogenous medaka ngn1 transcript. Further, we demonstrate that embryos from the transgenic line permit the non-destructive, real-time screening of ngn1 promoter-directed GFP expression in a 96-well format, enabling higher throughput studies of developmental neurotoxicants. This strain has been deposited with and maintained by the National BioResource Project and is available on request (http://www.shigen.nig.ac.jp/medaka/strainDetailAction.do?quickSearch=true&strainId=5660).