Identification of vascular disruptor compounds by analysis in zebrafish embryos and mouse embryonic endothelial cells

Abstract

To identify vascular disruptor compounds (VDCs), this study utilized an in vivo zebrafish embryo vascular model in conjunction with a mouse endothelial cell model to screen a subset of the U.S. Environmental Protection Agency (EPA) ToxCast Phase I chemical inventory. In zebrafish, 161 compounds were screened and 34 were identified by visual inspection as VDCs, of which 28 were confirmed as VDCs by quantitative image analysis. Testing of the zebrafish VDCs for their capacity to inhibit endothelial tube formation in the murine yolk-sac-derived endothelial cell line C166 identified 22 compounds that both disrupted zebrafish vascular development and murine endothelial in vitro tubulogenesis. Putative molecular targets for the VDCs were predicted using EPA’s Toxicological Prioritization Index tool and a VDC signature based on a proposed adverse outcome pathway for developmental vascular toxicity. In conclusion, our screening approach identified 22 novel VDCs, some of which were active at nanomolar concentrations

Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity

“…However, the compound is potent for developmental toxicity, causing severe malformations in the low micromolar range. A lack of angiogenic inhibition coupled with embryotoxicity was also noted in a separate zebrafish study following developmental exposure to 5HPP-33 [26]. These data indicate that 5HPP-33 may be developmentally toxic in zebrafish via mechanisms other than angiogenic inhibition.…”

“…These data indicate that 5HPP-33 may be developmentally toxic in zebrafish via mechanisms other than angiogenic inhibition. A recent publication showed that 5HPP-33 directly binds tubulin and disrupts microtubule dynamics during cellular mitosis [37] providing a mechanistic possibility for the widespread teratogenicity observed in the current study and McCollum et al [26]. Similar to 5HPP-33, a total of 14 of 29 chemicals that were negative for vascular toxicity caused malformations in zebrafish, including compounds like bisphenol AF, 4-nonylphenol, octyl gallate, and perfluorooctanesulfonic acid that were highly ranked by the predictive signature.…”

“…Data generated here were combined with angiogenesis toxicity data from McCollum et al ([26]; hit calls summarized in Supplemental Table S6). Hit calls were identical for seven compounds that were tested in the EPA and UH studies: bisphenol A, imazamox, oxytetracycline dehydrate, perfluorooctane sulfonic acid, pymetrozine, pyridaben, and triclosan (Supplemental Figure S3 and Supplemental Table S6).…”

“…Briefly, the log AC 50 values for each of the 821 in vitro HTS assays were compared with zebrafish toxicity results (EPA or UCD platforms). Additional zebrafish angiogenic toxicity from McCollum et al was also used in the analysis [26]. Results were evaluated using continuous (Student’s t -test and Pearson’s correlation test) and dichotomous (chi-squared test) statistical methodology, with the level of significance returned as p values.…”

Adverse Outcome Pathway on Aryl hydrogen receptor activation leading to early life stage mortality, via reduced VEGF

“…ToxCast highthroughput screening (HTS) data for 25 assays mapping to targets in embryonic vascular disruption signature [Knudsen and Kleinstreuer, 2011] were used to rank-order 1060 chemicals for their potential to disrupt vascular development. The predictivity of this signature is being evaluated in various angiogenesis assays, including tubulogenesis in endothelial cells from zebrafish, chick, mouse and human species Vargesson et al 2003;Knudsen et al 2016;McCollum et al 2016;Nguyen et al 2016].…”