PgmNr Z6185: Identification of skeletal disruptor compounds through in vivo screening in zebrafish.

Authors:
SAVINI THRIKAWALA ¹ ; Rachna Sachanandani ¹ ; Sanat Upadhyay ² ; Clifford Stephan ³ ; Nghi Nguyen ³ ; Mary Sobieski ³ ; Richard Judson ⁴ ; Jan-Åke Gustafsson ¹ ; Maria Bondesson ⁵

---

Institutes
1) Department of Biology and Biochemistry, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX; 2) Department of Computer Science, University of Houston, Houston, TX; 3) Center for Translational Cancer Research, Texas A&M Health Science Center, Institute of Biosciences and Technology, Houston, TX; 4) National Center for Computational Toxicology, Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, NC; 5) Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX.

Abstract:

Exposure to teratogens during embryonic development may lead to skeletal birth defects, including cranio-facial and limb malformations. We have used zebrafish as a model organism to identify skeletal disruptors. Transgenic zebrafish expressing fluorescent markers in bone or cartilage tissues were used to visually monitor the development of the cleithrum and craniofacial cartilage structures under normal and perturbed conditions. Next, transgenic skeletal embryos were used in a primary high-throughput screen of 309 compounds of the ToxCast phase I chemical inventory, primarily consisting of pesticides and antimicrobials. We found that exposure to 82 compounds perturbed normal development of the cleithrum. The compounds causing an effect in the primary screen were further analyzed across a wider dose range. From this we identified 38 skeletal disruptors; 29 of them caused malformations in the cleithrum and 9 caused a short cleithrum. By further analysis in cartilage transgenic fish, we found that exposure to some, but not all, of the compounds caused perturbed development of both skeleton and cartilage.

Univariate analysis was performed to identify ToxCast in vitro assays that significantly correlated with the identified in vivo zebrafish skeletal disruptors. Ten assays were identified and were used to create ToxPi profiles for the skeletal disruptors and rank them based on the AC₅₀ values of the ToxCast assays. A number of the corresponding molecular pathways to the ToxCast assays were further confirmed to be altered by exposures to skeletal disruptors in zebrafish. Additionally, RT-PCR was used to show that the expression of skeletal marker genes, such as Sox9 and Runx2, were affected by exposure to several of the skeletal disruptors. In conclusion, our results show that several environmental pollutants have skeletal disrupting capacity and that the developing zebrafish embryo is an efficient in vivo model that can be used for identification of skeletal disruptors and the molecular pathways that are affected by these chemicals.

ZFIN Genetics Index
1. runx2a
2. runx2b
3. sox9a
4. sox9b