Systems Modeling of Developmental Vascular Toxicity

Abstract

The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21^st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.

Keywords: 5HPP-33; Adverse outcome pathway; TNP-470; Tox21; ToxCast; angiogenesis.

Figure 1

Aop43 for embryonic vascular disruption (reproduced with permission from Knudsen and Kleinstreuer (2011) [5]).

Figure 2

Vascular disruption potential of 38 ToxCast™ chemicals selected to represent a range of pVDC scores from 0% (green) to 100% (red). ToxCast™ pVDC prediction scores are shown in column A, with 10 platform endpoints represented in columns B – K. Chemical endpoints in each of columns B - K were assigned a score of 1 (red) or zero (green), with cumulative in vitro scores (i.e., number of assay positives) indicated in column L. Gradient colors used in columns A and L correspond to pVDC scores (A) or cumulative in vitro scores (L). Black = cytotoxicity; white = not tested. Compared to total in vitro outcomes (L), the in silico (A) prediction sensitivity and specificity were 0.89 and 0.80, respectively; prediction accuracy was 87%

Figure 3

Transcriptomic analysis of rat whole embryo culture (WEC) at 4 days after exposure to angiogenesis inhibitors, 5HPP-33 (0 to 30 µM) or TNP-470 (0 to 25 µM). A) 9006 total differentially expressed genes (DEGs) at any exposure concentration compared to DMSO controls included 2831 that were significantly affected by both compounds. B) Self-organizing map (SOM) comprising all 9006 DEGs ranked 5HPP-33 (HPP) and TNP-470 (TNP) treatments by similarities in gene expression changes. Each of the 12 boxes represents a comparison between two treatments. SOM analysis identified a group of 464 genes (black box) that were similarly differentially expressed following exposure to 2.5 µM TNP-470 or 5 µM 5HPP-33. C) Fold-change comparison of 464 genes from region of interest (ROI) in part B. D) Pathway analysis exploring the effects of 2.5 µM TNP-470 and 5 µM 5HPP-33 on p53 signaling.

Figure 4

Updated pVDC signature ranking the 38 chemicals (circled; selected to represent a range of pVDC scores) among 1058 ToxCast™ Phase I and II chemicals. Each ToxPi slice represents a ToxCast™ molecular target included in the Aop43 model as described in Knudsen and Kleinstreuer (2011) [5]. 5HPP-33 and TNP-470 rank in the top 25% of chemicals.