Tert-butylphenol exposure alters cartilage and bone development in zebrafish

Phenolic antioxidants, such as 2,4-di-tert-butylphenol (2,4-DTBP), 2-tert-butyl phenol (2-BP), and 4-tert-butyl phenol (4-BP), are additives used in domestic water pipes, food packaging, paints, and other industrial products. As additives, they can leach from products and are frequently found in both environmental and human biological samples. Previous studies have demonstrated that 2,4-DTBP exposure can impair the differentiation of human iPS cells into somite- and sclerotome-like cells, and reduce key processes involved in osteoblast formation. Therefore, the goal of this study is to determine if 2-BP, 4-BP, 2,4-DTBP, and its metabolite 3,5-di-tert-butylcatechol (3,5-DTBC) impacts the development of cartilage and bone in vivo, using zebrafish as a model organism. Zebrafish embryos were exposed to increasing concentrations of each of the four chemicals from 1 h post fertilization (hpf) until 5 days post fertilization (dpf), and analyzed for markers of bone and cartilage development. At their highest concentrations tested, both 2-BP and 2,4-DTBP altered axial skeleton formation, with 76% and 61% of the zebrafish showing spinal curvatures, respectively. To corroborate these changes, the expression of marker transcripts were examined. 2-BP exposure reduced mRNA expression of the bone mineralization marker sparc by 1.6-fold. In contrast, 2,4-DTBP increased sparc transcript expression by 1.4-fold. All four compounds significantly upregulated sox9a, a chondrogenesis marker, between 1.4- to 5-fold. Changes in tail cartilage formation were noted using Alician blue staining, with 2,4-DTBP reducing width, length, and cartilage area of the tail, while 2-BP reduced the tail width but with increased the tail base, yielding a more straightened tail. Principle component analysis (PCA) demonstrated associations between sox9a, sparc, nrf2a, reactive oxygen species (ROS), and tail cartilage measurements, particularly in the 2,4-DTBP exposures, suggesting the involvement of nrf2a signaling in impairing cartilage formation. Overall, the study shows that each of the phenolic antioxidants differentially affects the development of bone and cartilage structures in zebrafish.