LINKING NANOPARTICLES TO EMBRYONIC DEFORMITIES: EXPLORING THE TERATOGENICITY OF ZINC OXIDE NANOPARTICLES

The exponential growth of nanotechnology has led to significant advancements in engineered nanoparticles (ENPs) between 1 and 100 nm in size, with zinc oxide nanoparticles (ZnO-NPs) playing a prominent role across various industries and applications. Particularly in biomedicine, ZnO-NPs have emerged as versatile tools, serving as anti- bacterial agents, drug and gene delivery platforms for cancer treatment, cellular imaging enhancers, and high-performance biosensors. For these reasons the aim of this study is to explore their potential toxicity on zebrafish early life stage using a combined in vivo and in-silico approach. In the first phase by SEM-EDS analysis, the ZnO-NPs purity was confirmed. After the Fish Embryo Acute Toxicity Tests according to OECD test guideline No. 236 (OECD, 2013) were performed. The embryos were exposed to five con centrations of ZnO-NPs: 50, 100, 150, 200 and 250 mg/L. At 96 hours, LC20 of about 58.201 mg/L and NOED of <50 mg/L, were determined. The most common sub-lethal alterations were pericardial and yolk edema, blood stasis, reduced blood circulation, reduced heartbeat, skeletal alterations and delayed hatching. Later, to further assess the toxicity of ZnO nanoparticles, oxidative stress was evaluated by quantifying lipid peroxidation using the thiobarbituric Acid Reactive Substances assay. The results showed as the treatment with ZnO-NPs led to a significant increase in lipid peroxidation in zebrafish larvae, as evidenced by the elevated levels of MDA induced by the treatment, indicating a low detoxification capacity of reactive oxygen species. To further confirm these findings, the gene expression of key antioxidant enzymes such as catalase (cat), superoxide dismutase (sod), and glutathione S-transferase (gstm) was also evaluated via RT-PCR. The results demonstrated a decrease in the expression of all the enzymes, suggesting that nanoparticles may interfere with the redox state of zebrafish larvae. Furthermore, since the oxidative stress is often associated with inflammation also key genes related to inflammation tnfalpha and il1beta were assayed. Our results showed a modulation of inflammation’s genes, particularly treatment induced a low downregulation of tnfalpha and an opposite regulation of il1beta which expression increased at very high levels. Finally, molecular docking and dynamics approach were applied to further explore any potential molecular interactions between ZnO- NPs and critical embryonic proteins, such as hatching enzyme ZHE1, and superoxide dismutase, SOD 1, enzyme. Results shown that ZnO-NPs interfered with both enzymes inhibiting those activities, and causing a delayed hatching of zebrafish embryos most probably through a multi-modal mechanism. The integration of in silico and in vivo assessments provides a more comprehensive evaluation of the potential risks associated with exposure to nano- materials, contributing to the fields of nanotoxicology and developmental biology. However, we are discussing preliminary results that require further testing and examination to fully understand the molecular mechanisms and causes of ZnO-NP toxicity.