High-throughput characterization of chemical-associated embryonic behavioral changes predicts teratogenic outcomes

Abstract

New strategies are needed to address the data gap between the bioactivity of chemicals in the environment versus existing hazard information. We address whether a high-throughput screening (HTS) system using a vertebrate organism (embryonic zebrafish) can characterize chemical-elicited behavioral responses at an early, 24 hours post-fertilization (hpf) stage that predict teratogenic consequences at a later developmental stage. The system was used to generate full concentration–response behavioral profiles at 24 hpf across 1060 ToxCast™ chemicals. Detailed, morphological evaluation of all individuals was performed as experimental follow-up at 5 days post-fertilization (dpf). Chemicals eliciting behavioral responses were also mapped against external HTS in vitro results to identify specific molecular targets and neurosignalling pathways. We found that, as an integrative measure of normal development, significant alterations in movement highlighted active chemicals representing several modes of action. These early behavioral responses were predictive for 17 specific developmental abnormalities and mortality measured at 5 dpf, often at lower (i.e., more potent) concentrations than those at which morphological effects were observed. Therefore, this system can provide rapid characterization of chemical-elicited behavioral responses at an early developmental stage that are predictive of observable adverse effects later in life.

Arsenic (III, V), indium (III), and gallium (III) toxicity to zebrafish embryos using a high-throughput multi-endpoint in vivo developmental and behavioral assay

“…Stimulation of a burst of tail flexions in the PMR with bright white light is a highly sensitive assay for which abnormal responses are highly predictive of later adverse developmental outcomes (Reif et al, 2015). An example of the 24 hpf zebrafish activity recorded in the PMR for In(III)-citrate is shown in Fig.…”

Assessment of the developmental and neurotoxicity of the mosquito control larvicide, pyriproxyfen, using embryonic zebrafish

“…This activity is driven by photoreceptors in the hindbrain and the response is typically refractory to a second light flash (Kokel and Peterson, 2008; Raftery et al, 2014; Reif et al, 2016). The assay thus requires keeping the developing embryos in the dark until 28 hpf when embryos are subjected to two flashes of a bright light with a 9 sec interval between pulses.…”

“…Animals dead or malformed at the 24 hpf timepoint were excluded from the behavior data sets. Statistical analysis was conducted as described in Reif et al (2016) (Reif et al, 2016). …”

Optimizing multi-dimensional high throughput screening using zebrafish

“…There are over 600 assays, and 800 assay endpoints that are evaluated to provide enough information to prioritize chemicals for further testing. As an effort to help with this initiative, the zebrafish model was used to screen the ToxCast Phase 1 and 2 library [2, 3, 10]. From these studies, there was a good concordance with historic animal data and amongst the different zebrafish assays, demonstrating this model is feasible for HTS screening.…”