Water Exposure Concentration (ug / mL)

1. Embryo production • Embryo dechorination • Exposure plating (media, embryo) • Assay • Time lapse imaging / detection 4 to 6 hours post fertilization (hpf) Left: embryo with chorion Right: dechorinated embryo Automated liquid handling Automated embryo handling One embryo per well 100 uL / well Microtiter Plate Equipment Well Characterized Test Materials 384 well 96 well Source: James E Hutchison, University of Oregon D. Tier 1: Accessing Behavior One embryo per well 100 uL / well Head Edema Shortened Snout Uninflated Gas Bladder Test Material Pericardial Edema Yolk Sac Edema Zebrafish (Danio rerio) Control • Robust in vivo model organism platform to evaluate nanomaterial biological interactions • Vertebrate animal homologous to humans, sequenced genome, sensitive at multiple levels • Compatible with high throughput screening, automation, pathway, and mechanistic studies • The NANO revolution has begun – get ready Heart Positive Charge Negative Charge Neutral Charge Percent of Total Number (%) with Biological Effect Hazard Identification Tanguay Laboratory Funding Agency TMAT 12-24 Percent Embryos Effected Air Force Research Laboratory; # FA8650-05-1-5014 EPA RD-833320 NIEHS P3000210, ES016896 MES 12-48 no detect MEEE Nanomaterials Dose Response Exposure 6 24 48 72 96 120 hpf Hutchison, J; Zaikova, T; University of Oregon; Gold Orr, G; Pacific Northwest National Laboratory; Silica Naik, R; Air Force Research Laboratory; Quantum Dot Water Exposure Concentration (ug / mL) Health Risk Experiment 400 400 Nanomaterial Source: Hutchison, J, UO Experiment: Truong, L, OSU TMAT – N,N,N-trimethylammoniumethanethiol MES – 2-mercaptoethanesulfonate MEEE - [mercaptoethoxy(ethoxy)]ethanol 300 300 Working Groups 200 200 Gold (Au) Nanograms / Embryo National Cancer Institute (NCI) Biomedical Informatics Grid (caBIG) Nanotechnology Office of the Secretary of Defense (OSD) Nanomaterials ESOH The Oregon Nanoscience and Microtechnologies Institute (ONAMI) Safer Nanomaterials and Nanomanufacturing Initiative (SNNI) 100 100 0 0 24 hpf 48 hpf 24 hpf 48 hpf 24 hpf 48 hpf 24 hpf 48 hpf 24 hpf 48 hpf 24 hpf 48 hpf Water Exposure Concentration (ug / mL) Nanomaterial Hazard Identification: The Zebrafish Model for Rapid Material Testing Maj Joseph A. Fisher, 349th Medical Squadron, Travis AFB, CA Dr. Robert L. Tanguay, Oregon State University, Corvallis, OR ABSTRACT Force Health Protection is facing a new challenge both in-garrison and in deployed operations as the nanotechnology revolution begins. The National Science Foundation predicts the period from 2011-2020 will result in fundamentally new products based on nanomaterials. These chemical biophysical nanometer scale (i.e., 1 x 10-9 meters) materials may bring new or increased hazard to humans and the environment, and the uncertainty surrounding their risk to biological and environmental health needs to be investigated. Health risk can be defined as a function of hazard and exposure, and an understanding of the hazard and exposure of these materials is important in order to minimize health risk. Products utilizing nanoscale materials will become ubiquitous throughout commerce in the coming years and regulatory oversight and reporting in the EU and the US is moving forward. The development of the zebrafish (Danio rerio) model for rapid material testing bridges a gap in toxicology testing between in vitro cell culture models and in vivo mammalian models. The anatomy, physiology, and genomics of the zebrafish are highly homologous to humans, and these similarities are just beginning to be exploited by research communities. Being a whole animal vertebrate organism, zebrafish allow for great flexibility in conducting experimental assays to identify nanomaterial exposure effects in morphology, physiology, behavior, and distribution. This research presents an overview of the issues surrounding nanomaterial health risk and provides testing results in order to demonstrate the utility of the zebrafish model in answering nanomaterial bio-compatibility research questions. AUTOMATION MATERIALS AND METHODS A. Precisely engineered Gold (Au) Nanomaterials B. In Vivo Zebrafish Tier 1: Morphology, Physiology, Behavior Tier 2: Cellular Targets and Distribution Tier 3: Molecular Expression 28 °C (83 °F) Embryo production and dechorination is facilitated by high capacity high throughput systems. Liquid handlers and robots facilitate experiment setup. Imaging systems support automated observation. C. Tier 1: Accessing Morphology CONCLUSION A. Precisely engineered (physiochemical and structural) materials are needed in order to determine biological effect. B. In vivo Zebrafish provide a tiered testing scheme to analyze materials. Zebrafish are screened for response (effect) during 1-5 days post fertilization (dpf). C. 20 morphological parameters are evaluated. D. 3 behavior parameters (spontaneous movement, swimming, and touch response) are evaluated. RESULTS Exposure assessment up to 120 hpf – 3 ligand types and 2 sizes Statically exposed to gold (Au) naomaterials from 6 hpf We need to test materials “better – cheaper – faster” in order to keep up with the large number of new nanomaterials expected in the near future. A. Tier 1: Morphology B. Exposure Time Period Sensitivity INTRODUCTION ACKNOWLEDGEMENT D. Distribution (uptake) C. Tier 1: Behavior • Testing Platforms • In silico (virtual screening, models) • In vitro (primary/finite and continuous cell cultures) • In vivo (whole animals study): • Mouse / rat • Fish / amphibian • Fly / worm - invertebrate • Clinical trials • Epidemiology Nanomaterial Source: Hutchison, J, UO; Experiment: Truong, L, OSU TMAT - N,N,N-trimethylammoniumethanethiol MES - 2-mercaptoethanesulfonate MEEE - [mercaptoethoxy(ethoxy)]ethanol A. Morphology assessment detects lethal effect with TMAT, sub-lethal effect with MES, and “no detect” with MEEE. B. The exposure time period sensitivity varies with material. C. Behavior assessment detects TMAT and MES effect and “no detect” with MEEE. D. Distribution (uptake) occurs with all materials. Sinnhuber Aquatic Research Laboratory Oregon State University, Corvallis, OR A Paradigm shift in testing is needed. 2011 Air Force Medical Service (AFMS) Medical Research Symposium