Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster

Summary

We describe a detailed protocol for evaluating the toxicological profiles of zinc oxide nanoparticles (ZnO NPs) in particular, the type of cell death in human MRC5 lung fibroblasts and ROS formation in the fruit fly Drosophila.

Abstract

Zinc oxide nanoparticles (ZnO NPs) have a wide range of applications, but the number of reports on ZnO NP-associated toxicity has grown rapidly in recent years. However, studies that elucidate the underlying mechanisms for ZnO NP-induced toxicity are scanty. We determined the toxicity profiles of ZnO NPs using both in vitro and in vivo experimental models. A significant decrease in cell viability was observed in ZnO NP-exposed MRC5 lung fibroblasts, showing that ZnO NPs exert cytotoxic effects. Similarly, interestingly, gut exposed to ZnO NPs exhibited a dramatic increase in reactive oxygen species levels (ROS) in the fruit fly Drosophila. More in-depth studies are required to establish a risk assessment for the increased usage of ZnO NPs by consumers.

Introduction

Nanotechnology refers to the application of nanosized materials that are used across all scientific fields, including medicine, materials science, and biochemistry. For instance, ZnO NPs which are known for their ultraviolet scattering, chemical sensing, and anti-microbial properties, as well as high electrical conductivity, are utilized in the production of various consumer products such as food packaging, cosmetics, textiles, rubbers, batteries, catalyst for automobile tail gas treatment, and biomedical-related applications^1,2,3.

However, the bur....

Protocol

1. Fluorescence Activated Cell Sorting (FACS) Analysis on Lived/Fixed cells

1. Sonicate ZnO NPs in suspension for 15 min.
2. Prepare ZnO NPs at various concentrations (e.g., 0, 10, 25, 50,100 and 200 µg/mL) using 1 mg/mL ZnO NP stock solution for the treatment of cultured cells.
3. Seed MRC5 human lung fibroblasts (1 x 10⁵ cells/well) onto a 6-well culture plate a day in advance, and then treat the cells with 2 mL of ZnO NPs (in triplicates) for 8 h, 16 h, and 24 h.
4. At eac.......

Discussion

In order to assess if ZnO NP can induce apoptosis in MRC5 fibroblasts, we use flow cytometry to distinguish the cells from necrotic or apoptotic cell death. In normal live cells, phosphatidylserine (PS) is localized at the cell membrane. If apoptosis occurs, PS is translocated to the extracellular leaflet of the plasma membrane, allowing the binding of Annexin V labeled with fluorescein (FITC Annexin V)²⁹. On the other hand, the red-fluorescent propidium iodide (PI), a nucleic acid binding dye, is.......

Materials

Name	Company	Catalog Number	Comments
15% Methyl 4-Hydroxybenzoate	Sigma Aldrich
4% Paraformaldehyde	Sigma Aldrich	P6148
Bacto Agar	BD biosciences
cncCK6/TM3, Sb			a gift from Dr. Kerppola T
cornmeal, glucose, yeast brewer	Sigma Aldrich
CyAn ADP with Summit Software	DAKO		https://flow.usc.edu/files/2014/07/BC-Cyan-ADP-User-Guide-2016.pdf
Dihydroethidium (Hydroethidine)	Thermo Fisher Scientific	D11347
FITC Annexin V Apoptosis Detection Kit I	BD biosciences	556547
Fluorescent microscope	Olympus
Glucolin	Supermarket
Image J software	NIH
MRC5 human lung fibroblast	ATCC	CCL-171
Schneider’s Drosophila medium	Thermo Fisher Scientific	21720-024
vectashield antifade mounting medium with DAPI	Vector Laboratories	H-1200
wild- type Canton-S; Sod2N308/CyO	NIG-FLY
Zinc Oxide Nanoparticles	Sigma Aldrich	721077	Refer Sheet 2