Applications of Zebrafish Embryo Models to Predict Developmental Toxicity for Agrochemical Product Development.

The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.

Triazole fungicides induce adipogenesis and repress osteoblastogenesis in zebrafish.

Triazoles are a major group of azole fungicides commonly used in agriculture, and veterinary and human medicine. Maternal exposure to certain triazole antifungal medication causes congenital malformations, including skeletal malformations. We hypothesized that triazoles used as pesticides in agriculture also pose a risk of causing skeletal malformations in developing embryos. In this study, teratogenic effects of three commonly used triazoles, cyproconazole, paclobutrazol, and triadimenol, were investigated in zebrafish, Danio rerio. Exposure to the triazole fungicides caused bone and cartilage malformations in developing zebrafish larvae. Data from whole-embryo transcriptomics with cyproconazole suggested that exposure to this compound induces adipogenesis while repressing skeletal development. Confirming this finding, the expression of selected bone and cartilage marker genes were significantly downregulated with triazoles exposure as determined by quantitative PCR. The expression of selected adipogenic genes was upregulated by the triazoles. Furthermore, exposure to each of the three triazoles induced adipogenesis and lipid droplet formation in vitro in 3T3-L1 pre-adipocyte cells. In vivo in zebrafish larvae, cyproconazole exposure caused lipid accumulation. These results suggest that exposure to triazoles promotes adipogenesis at the expense of skeletal development, and thus they expand the chemical group of bona fide bone to fat switchers.

E-cigarette vaping liquids and the flavoring chemical cinnamaldehyde perturb bone, cartilage and vascular development in zebrafish embryos.

As electronic cigarettes (e-cigarettes) become increasingly popular smoking devices, there is an increased risk for unintended exposure to e-cigarette liquids through improper disposal resulting in leaching into the environment, third hand vapor exposure through air, or embryonic exposure through maternal vaping. Thus, the safety of e-cigarettes for wildlife and developing embryos need to be thoroughly investigated. We examined perturbations in zebrafish embryonic development after exposures to two cinnamon flavored vaping liquids (with 12 mg/ml nicotine and without nicotine) for e-cigarettes from two different vendors, as well as the flavoring chemical cinnamaldehyde. We focused on the effects of the vaping liquids on hatching success and bone, cartilage and blood vessel development in 3-4 days old transgenic zebrafish larvae. We found that exposures to both of the vaping liquids perturbed the development of the cleithrum and craniofacial cartilage. Exposure to the liquids further caused non-overlapping and partially or completely missing intersegmental vessels. Hatching success was also reduced. Exposure to pure cinnamaldehyde replicated the effects of the vaping liquids with a 50% effect concentration (EC50) of 34-41 µM. Quantification of the amount of cinnamaldehyde in the vaping liquids by mass spectrometry revealed EC50s around 10-40 times lower than for pure cinnamaldehyde, suggesting that additional compounds or metabolites present in the vaping liquids mediate toxicity. Presence of nicotine in one of the vaping liquids decreased its EC50s about two fold compared to the liquid without nicotine. Exposure to the humectants propylene glycol and vegetable glycerin did not affect the vascular, cartilage or bone development in zebrafish embryos. In conclusion, our study shows that exposure to cinnamaldehyde containing vaping liquids causes severe tissue-specific defects in developing embryos.