Investigation of Chondrogenesis, Myogenesis, and Osteogenesis After 2,4-DTBP, 2-BP, 4-BP, 3,5-DTBC Exposure in Zebrafish

Synthetic phenolic antioxidants (SPAs) are used in personal care products, food packaging, and water pipes, common SPAs include 2,4-di-tert-butylphenol (2,4-DTBP), 2-tert-butyl phenol (2-BP), and 4-tert-butyl phenol (4-BP). These compounds are detected in the environment and in human biological samples. 2,4-DTBP has been detected in cord plasma, placenta, and breast milk samples. This drives the concern regarding developmental toxicity. In human iPS cells 2,4-DTBP impairs differentiation into somites and osteoblasts. Other SPAs, like 2,6-di-tert-butyl-hydroxytoluene caused increased rates of malformations including yolk and pericardial edema, and spinal curvature in 5-day post fertilization (dpf) zebrafish. SPAs can disrupt levels of reactive oxygen species (ROS) which are crucial for differentiation of osteoblasts and chondrocytes. This study aimed to investigate if 2,4-DTBP, 3,5-DTBC, 2-BP, and 4-BP effects development of bone, cartilage, and muscle in zebrafish. Zebrafish were exposed to 2,4-DTBP, 3,5-DTBC, 2-BP, or 4-BP from 1 hour post fertilization to 5 dpf, mortality, hatching, and morphology were assessed. Gene expression was measured and cartilage and bone development was assessed with Alcian blue and Alizarin red staining. 2-BP and 3,5-DTBC significantly decreased expression of sparc, a gene required for bone formation. 3,5-DTBC and 4-BP significantly increased expression sox9a, a transcript required for cartilage formation and myod1, a gene involved in muscle development. Staining analysis revealed 2,4-DTBP and 2-BP significantly reduced length of cartilage structures and the distance between structures. 3,5-DTBC and 4-BP increased both distance and angle of cartilage structures. This study shows that 2,4-DTBP, 3,5-DTBC, 2-BP, and 4-BP differentially alter early development of zebrafish larvae.