Mechanistic insight into ROS and neutral lipid alteration induced toxicity in the human model with fins (Danio rerio) by industrially synthesized titanium dioxide nanoparticles

Abstract

The toxicological impact of TiO₂ nanoparticles on the environment and human health has been extensively studied in the last few decades, but the mechanistic details were unknown. In this study, we evaluated the impact of industrially prepared TiO₂ nanoparticles on the biological system using zebrafish embryo as an in vivo model. The industrial synthesis of TiO₂ nanoparticles was mimicked on the lab scale using the high energy ball milling (HEBM) method by milling bulk TiO₂ particles for 5 h, 10 h, and 15 h in an ambient environment. The physiochemical properties were characterized by standard methods like field emission scanning electron microscopy (FESEM), dynamic light scattering (DLS), X-ray diffraction (XRD) and UV-Visible spectroscopy. In vivo cytotoxicity was assessed on zebrafish embryos by the evaluation of their mortality rate and hatching rate. Experimental and computational analysis of reactive oxygen species (ROS) induction, apoptosis, and neutral lipid alteration was done to study the effects on the cellular level of zebrafish larvae. The analysis depicted the change in size and surface charge of TiO₂ nanoparticles with respect to the increase in milling time. In silico investigations revealed the significant role of ROS quenching and altered neutral lipid accumulation functionalised by the molecular interaction of respective metabolic proteins in the cytotoxicity of TiO₂ nanoparticles with zebrafish embryos. The results reveal the hidden effect of industrially synthesized TiO₂ nanoparticle exposure on the alteration of lipid accumulation and ROS in developing zebrafish embryos. Moreover, the assessment provided a detailed mechanistic analysis of in vivo cytotoxicity at the molecular level.

Fig. 1. Characterization of bulk, 5 h, 10 h and 15 h milled TiO₂ nanoparticles prepared by the HEBM method. FESEM images of (A) bulk, (B) 5 h nano TiO₂ (C) 10 h nano TiO₂ (D) 15 h nanoTiO₂. (E) XRD spectra of nanoparticles.

Fig. 2. Graphical representation of hatching rate and viability rate of zebrafish embryo treated with low (50 μg mL^–1) and high (250 μg mL^–1) concentrations of bulk TiO₂ and nanoparticles. (a), (b) The hatching rate at low and high concentration, respectively; (c), (d) the viability percentage at low and high concentration, respectively. Percentages were determined for a group of 20 embryos. The values represent the mean ± SD of three independent experiments. *P < 0.05 denotes the significant change from untreated control, bulk and 7 h exposed embryos as obtained from two way ANOVA analysis; * represents the degree of significance.

Fig. 3. Morphological and anatomical changes in zebrafish embryo treated with low (50 μg mL^–1) and high (250 μg mL^–1) concentrations of bulk TiO₂ and nanoparticles. The bright field image was taken after washing twice with HF buffer. The red arrows indicate the deformation in embryos (deformation in chorion and yolk sac in 48 hpf, and deformation in the tail, notochord and heart development in 96 hpf). Scale bar represents 1000 nm.

Fig. 4. A. Molecular interaction of he1a protein with TiO₂, visualized using Discovery Studio Visualizer, showing the mode of interaction with residues. B. Pathway showing the TiO₂ interaction mechanism through f2 catalyst to he1a.

Fig. 5. Schematic representation of the effect of TiO₂ nanoparticles on zebrafish embryo hatching.

Fig. 6. Oxidative stress analysis; ROS measurement of zebrafish embryos treated for 24 h and 48 h at low (50 mg L^–1) and high (250 mg L^–1) concentrations of bulk TiO₂ and nanoparticles as determined by flow cytometry. Fluorescence intensity of cells shifted towards the left at (A) 48 hpf and (B) 96 hpf in accordance with the milling time. (C) Molecular docking analyses of sod1 with TiO₂ nanoparticles showing interacting residues using LigPlot+ and Discovery Studio Visualizer.

Fig. 7. Fluorescent images of zebrafish larvae stained with Lipidtox dye treated with TiO₂ nanoparticles at concentrations of 50 μg mL^–1 and 250 μg mL^–1, presenting alterations in neutral lipids in different tissues. Neutral lipid accumulation alteration was found to vary with the size and concentration of the nanoparticles.

Fig. 8. Molecular docking analysis. (A) Interaction of apoa1a with TiO₂ nanoparticles showing interacting residues using LigPlot+ and Discovery Studio Visualizer. (B) Protein–protein interaction of MTTP and apoa1a proteins showing the site of interaction using Chimera. (C) Interaction of tp53 with TiO₂ nanoparticles, showing interacting residues using LigPlot+ and Discovery Studio Visualizer.

Fig. 9. Fluorescent images of zebrafish larvae stained with acridine orange (AO), treated with TiO₂ nanoparticles at concentrations of 50 μg mL^–1 and 250 μg mL^–1, presenting apoptosis in the head and tail tissues. Arrows indicate the sites of apoptosis. Apoptosis was found to vary with the size and concentration of the nanoparticles.

Fig. 10. Pathway showing the TiO₂ interaction mechanism involving sod1, apoa1a, MTTP and tp53 proteins derived from STITCH and analyzed using Cytoscape.

Fig. 11. Schematic representation of the effects of TiO₂ nanoparticles on oxidative stress, neutral lipid metabolism and apoptosis.