Adverse Outcome Pathways: A framework for Identifying Chemical Mode of Action During Zebrafish Embryogenesis

Human health and ecological risk assessors are currently facing increased demands and public pressure to evaluate the potential risk of hundreds to thousands of chemicals based on limited toxicity data. Therefore, scientifically sound models are needed to predict toxicity without sole reliance on conventional whole-organism (vertebrate) bioassays. To help address this challenge, adverse outcome pathways (AOPs) have recently been proposed as conceptual tools to help link direct, molecular-level initiating events to adverse outcomes at higher levels of biological organization. Additionally, using AOPs as frameworks, high-throughput screening (HTS) and high-content screening (HCS) assays have been recently proposed as key components of a tiered regulatory toxicity testing strategy that enables screening and prioritization of chemicals for further testing using conventional bioassays. Therefore, using zebrafish embryonic development as a model, the overall research aims of this dissertation were to 1) rely on paraoxon - the active metabolite of the organophosphorus (OP) insecticide parathion - as a reference acetylcholinesterase (AChE) inhibitor to examine whether the common molecular initiating event (AChE inhibition) was associated with adverse neurological effects at higher levels of biological organization and 2) develop and optimize a HCS assay to identify chemicals impacting cardiovascular function and potentially serve as a platform for guiding AOP discovery and development. Data presented within Chapter 1 suggests that (1) normal AChE activity is not required for secondary motoneuron development and (2) spontaneous tail contractions at 26 hours postfertilization (hpf) are sensitive to paraoxon exposure, an effect that may be independent of AChE inhibition. Data presented within Chapter 2 suggests that, compared to existing zebrafish-based assays, our HCS assay provides a comprehensive chemical screening and AOP discovery platform with 1) increased sample sizes; 2) broad concentration-response format; and 3) the ability to identify chemicals that target cardiovascular function at non-teratogenic concentrations. Overall, this dissertation highlights the scientific and logistical challenges in developing quantitative AOPs to support chemical screening and prioritization strategies. Nonetheless, qualitative AOPs can play a role in identification of HTS/HCS assays that capture key events along an AOP, and results of HTS/HCS-based chemical screens can be used to facilitate development of current and new AOPs

Tidal freshwater ecosystems: bibliography

Tidal freshwater ecosystems represent an important transition zone between saline reaches of estuaries and non-tidal riverine environments. Tidal freshwater systems are distributed worldwide, but have been intensively studied in only a few geographic regions, such as the U.S. east coast and western Europe. Typically, tidal freshwater systems are characterized by high physical stress due to sediment instability and tidal action, which results in low species diversity. However, a number of anadromous and resident fish species utilize tidal freshwater reaches of estuaries as a spawning and nursery area, including economically significant species such as striped bass, American shad, and Atlantic sturgeon. Tidal freshwater marshes are a unique wetland community type, and are utilized extensively by migratory and wading bird species. Much of the research conducted in tidal freshwater ecosystems, particularly in the mid-Atlantic region of the U.S., has focused on the ecology of tidal freshwater marshes. The existing literature on tidal freshwater ecosystems is scattered among numerous technical journals spanning a variety of scientific disciplines. A considerable body of gray literature, in the form of agency and institutional reports, is available. We have included both the primary and grey literature in our compilation, and indexed the body of work by author and subject. We hope that this resource will benefit current and future scientists and resource managers working in tidal freshwater ecosystems

Adapting the Implicit Association Test to Health Professions Education May Lead to Improving American Indian Health

The Implicit Association Test (IAT) has been supported as a valid means for demonstrating implicit biases in racial/ethnic studies; however, adaption of the IAT for evaluation of unconscious biases against American Indians has not previously been reported. The authors conducted a study addressing validity of the IAT for measurement of implicit bias against American Indians in a health education setting, its value in providing information not revealed by explicit measures, and the association between demographic characteristics of health professions student groups and their IAT scores. The paper-format IAT instrument designed was piloted at an Oklahoma institution with health professions students. The American Indian participant group demonstrated an implicit preference for American Indian over Caucasian surnames by faster association of American Indian surnames with good versus bad attribute exemplars. Demonstration of predictable implicit response patterns that varied from explicit responses provided evidence for the value of adaptation of an IAT to evaluate American Indian bias. American Indian implicit bias research with health care professionals and students using the IAT may provide a valuable means for reaching the ultimate goal of improving health care for American Indians and thereby improving the health of American Indian tribal communities

Strong Zonation of Benthic Communities Across a Tidal Freshwater Height Gradient

Trade-offs associated with environmental gradients generate patterns of diversity and govern community organisation in a landscape. In freshwaters, benthic community structure is driven by trade-offs along generally orthogonal gradients of habitat permanence and predation—where ephemeral systems are physiologically harsh because of drying stress, but inhabitants are less likely to be under the intense predation pressure of more permanent waterbodies. However, in tidal freshwaters, these two stressors are compounding, and the trade-offs associated with them are decoupled. 2. We investigated benthic community structure in a tidal freshwater habitat. These communities experience a suite of conditions atypical for a freshwater habitat: twice-daily drying; and high predation pressure by mobile fishes. We compared benthic communities at three tidal heights (low, mid, high) and contrasted these with nearby non-tidal freshwaters that varied in their hydrology (permanent, temporary). 3. We found that communities were more strongly differentiated in tidal freshwater habitats than between permanent and temporary inland freshwaters, which was surprising given the high interconnectedness and condensed longitudinal scale of tidal habitats. The differentiation of communities in tidal habitats was probably driven by the combined gradients of desiccation risk at low tide and intense predation by fish at high tide—a combination of pressures that are novel for the evolutionary history of the regional freshwater invertebrate fauna. 4. Our study provides evidence that environmental gradients can produce stronger patterns of community zonation than would be predicted for habitats that are spatially contiguous and have little or no dispersal limitation. These results give insight into how communities might respond if drivers of community structure are altered or reorganised from their regional or evolutionary norms

High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism-focused investigations within zebrafish and mammalian models

High-Content Screening Assay for Identification of Chemicals Impacting Spontaneous Activity in Zebrafish Embryos

Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (<i>fli1:egfp</i>) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration–response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3–100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA’s ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoateboth potent avermectinsabolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants

High-Content Screening Assay for Identification of Chemicals Impacting Cardiovascular Function in Zebrafish Embryos

Targeted assays are needed to better evaluate effects of chemicals on organogenesis and begin classification of chemicals by toxicologically relevant modes-of-action. Using transgenic zebrafish (<i>fli1:egfp</i>) that stably express eGFP within vascular endothelial cells, we have developed and optimized a 384-well-based high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular function at sublethal, nonteratogenic concentrations. Following static exposure of one embryo per well from 5 to 72 h postfertilization (hpf), automated image acquisition procedures and custom image analysis protocols are used to quantify body length, circulation, heart rate, pericardial area (a biomarker for cardiac looping defects), and intersegmental vessel area within freshly hatched live embryos. After optimizing 72 hpf anesthetization procedures, we evaluated each end point across four independent control plates containing 384 initial embryos per plate. Survival and imaging success rates across these plates ranged from 93 to 99% and 42 to 74%, respectively. Criteria were then defined for assay success and analysis of treatments, and 10 chemicals were screened for targeted effects on cardiovascular function. Compared to existing zebrafish-based assays, this method provides a comprehensive discovery platform with (1) increased sample sizes; (2) broad concentration–response format; and (3) the ability to identify chemicals that target cardiovascular function at nonteratogenic concentrations