Multidimensional in vivo hazard assessment using zebrafish

Abstract

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies.

Keywords: Tox21; ToxCast.; developmental; high-throughput screening.

FIG. 1.

Experimental approach for screening developmental and neurotoxicity for 1078 ToxCast chemicals. Embryos were dechorionated at 4 hours post fertilization (hpf), and plated at 6 using automated embryo placement systems. After which, 1 chemical was added to 2 plates, at 6 concentrations (0.0064–64μM, 10-fold serial dilution), n = 16 per plate × 2 plates. The embryos were statically exposed to chemical until 120 hpf. At 24 hpf, photomotor response data were collected, and 4 developmental endpoints were assessed. At 120 hpf, larval behavior and 18 morphological and behavioral endpoints were assessed. Abbreviation: DMSO, dimethyl sulfoxide.

FIG. 2.

Estimation of LEL from concentration-response data. The concentration-response plots for 3 endpoints {MORT, YSE, AXIS} are shown for the chemical ziram. The horizontal axis shows the 6 concentrations tested (0 = control). For each concentration, the incidence (number of responses = 1) across all 32 replicates is plotted as stacked points. The points exceeding the binomial significance threshold for each endpoint are colored red. Abbreviation: LEL, lowest effect level.

FIG. 3.

Bicluster heat map of chemicals with at least 1 hit in a specific developmental endpoint. The responses were clustered using Ward’s method by Euclidean distance between LELs. The heatmap is colored so that increasingly potent LEL responses are darker shades of blue, with inactive responses having no color. Many of the 304 chemicals hitting at least 1 specific endpoint also caused embryonic lethality, indicated by the black sidebar. Abbreviation: LEL, lowest effect level.

FIG. 4.

Correlation between endpoints. The 2 halves of the endpoint-endpoint correlation plot show the linear correlation between -log(LEL) results. The upper panel shows the correlation, r, with increasingly large font as the value increases. The lower panel plots the results summarized in the upper panel. Abbreviation: LEL, lowest effect level.

FIG. 5.

Sources of variation visualized by PCA. PCA was performed on the LEL matrix of all 1060 chemicals. Plotting symbols annotate chemicals into activity categories: mortality only (empty triangle), mortality plus specific endpoint(s) (black triangle with red filling), specific endpoint(s) only (red solid circle), or inactive (hollow red circle). The points are scaled according to how many specific endpoint LELs are associated with each chemical. Abbreviations: LEL, lowest effect level; PCA, principal components analysis.

FIG. 6.

Analysis of lowest effect level (LEL) by endpoint. The minimum LEL for each condition was computed for each concentration (plotted in order of increasing 10-fold potency, from 64 to 0.0064μM, notated 1–5) and endpoint (18 total). The vertical axis counts the number of chemicals meeting each concentration-condition-endpoint. Four LEL conditions were evaluated: mortality only (empty triangle), a specific endpoint LEL then mortality at a higher dose (red empty triangle), a specific endpoint only (red circle), or mortality and a specific endpoint occurring (black triangle with red filling).

FIG. 7.

Histogram of Log K _ow by biological activity and Log K _ow. Separate histograms are plotted for chemicals classified as “Hits” (pink) or “Negatives” (blue), with Log K _ow along the horizontal axis. The purple shading represents overlap between the 2 distributions.