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 burgeoning applications of ZnO NP-based products, leading to increased human exposure to ZnO NPs, have raised concerns on their potential adverse effects on human health. A number of in vitro cellular studies have demonstrated that ZnO NPs can induce oxidative stress, autophagy-related cytotoxicity, inflammation, and genotoxicity^4,5,6,7,8. Notably, the toxicity of ZnO NPs is assumed to be caused by the dissolution of Zn to free Zn²⁺ ions, as well as the surface reactivity of ZnO, resulting in the cellular ionic and metabolic imbalances that are linked with impaired ionic homeostasis and an inhibition of ion transportation^4,7,9,10. Importantly, studies have shown that the generation of reactive oxygen species (ROS) is one of the primary mechanisms underlying ZnO NPs-associated toxicity. Insufficient anti-oxidative activity following ROS insult has been shown to be responsible for eliciting the cytotoxicity and DNA damage⁹. The toxic effects of ZnO NPs have also been reported in animal models, including rodent¹, zebrafish^11,12, as well as the invertebrate Drosophila¹³.

Drosophila serves as a well-established alternative animal model for toxicity screening of chemical entities and nanomaterials (NMs)^14,15. Importantly, there are high levels of genetic and physiological similarity between human and Drosophila that justifies the use of Drosophila as an in vivo model for evaluating biological responses to environmental contaminants such as NMs¹⁶. Furthermore, there are many advantages of using Drosophila due to its small size, short lifespan, genetic amenability, and easy and cost-effective maintenance. Moreover, Drosophila has been widely adopted for the study of genetics, molecular and developmental biology, ever since its full genome was fully sequenced years ago back in 2000, therefore making it suitable for a variety of high-throughput screening and for tackling unresolved biological questions^{17,18,19,20,21}. In recent years, a number of studies related to immunotoxicity using different types of NPs in Drosophila have been reported^15,22,23,24. This fundamental new knowledge obtained from the studies using Drosophila has helped to provide more insights into our understanding of nanotoxicology.

ROS is a well-known culprit for cytotoxicity and genotoxicity caused by NPs, in particular, metal-based NPs²⁵. ROS are oxygen-containing chemical species with higher reactive properties than molecular oxygen. Free radicals such as superoxide radical (O₂^-) and even, non-radical molecules such as hydrogen peroxide (H₂O₂) can act as ROS. Under normal physiological condition, they are required to maintain cellular homeostasis²⁶, however, excessive ROS due to overproduction or dysregulation of the antioxidant defense system can cause oxidative stress, leading to damage to proteins, lipids and deoxyribonucleic acid (DNA)²⁷. For instance, as ROS levels increase and glutathione (GSH) level decreases concomitantly, disruption of adenosine triphosphate (ATP) synthesis takes place and lactate dehydrogenase (LDH) level increases in the medium, culminating in cell death²⁷.

Here, we provide protocols for performing cellular and genetic analyses using cultured mammalian cells and Drosophila to determine the potential adverse effects of ZnO NPs. An overview of the method used for the toxicity study of ZnO NPs is shown in Figure 1.

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 each time point, collect the cells by centrifuging at 300 x g for 5 min.
5. Wash the cell pellets twice with phosphate buffered saline (PBS).
6. Resuspend the cells with 1x binding buffer, which is composed of 0.1 M HEPES/sodium hydroxide (NaOH), 1.4 M sodium chloride (NaCl), and 25 mM calcium chloride (CaCl₂), at a concentration of 1 x 10⁵ cells per 100 µL.
7. Add 5 µL of Fluorescein isothiocyanate (FITC) Annexin V stain and 5 µL of propidium iodide (PI) DNA stain, and incubate the cells for 15 min at room temperature (RT; 25 °C) in dark.
8. Top up the samples with an additional 400 µL of 1x binding buffer before sorting the cells by flow cytometry. A minimum of 10,000 cells is analyzed for each sample.
9. Tabulate the bar chart using the median intensity obtained.

2. Exposure of ZnO NPs to Drosophila

1. Add 1 mL of nanoparticles at different concentrations into vials, followed by 9 mL of fly food to make a final concentration of 0.1 mg/mL, 0.25 mg/mL or 0.5 mg/mL ZnO NPs.
2. Mix the nanoparticles with food thoroughly in the vials using the pipette.
3. Allow fly food containing ZnO NPs to cool for at least 2-3 h before use.
4. Introduce adult male and female flies into the vials for 5 days, and allow them to mate and lay eggs (which appear as white spots) on the surface of the food.
5. Remove the parental flies, and allow the eggs to undergo further development, which consists of 4 different developmental stages (embryonic, larval, pupal and adult stage).

3. Dissection of Fly

1. Collect late 3^rd instar larvae from the wall of the vials for analyses. Freshly laid eggs normally develop into late 3^rd instar larvae after 72-120 h at RT.
2. Clean the dissection dish and fill up the well with dissection medium/PBS.
3. Dissect the larvae (late 3^rd instar) under the stereomicroscope, using a pair of forceps.
4. Use the tip of the forceps to make a tiny hole and break open the cuticle layer of the larvae. Carefully pull out the gut and place it into a 1.5 mL microcentrifuge tube containing Schneider's Drosophila medium, prior to the fixing step using 1 mL of 4% paraformaldehyde (PF).
5. 3.5Fix the gut in PF for 10 min at RT, for subsequent experiments, such as immunostaining.

4. ROS Detection Using Dihydroethidium (DHE) Staining

1. Treat larvae with various concentrations of ZnO NPs as described in step 2.1.
2. Following the dissection of the gut from 3^rd instar larvae as described under section 3, incubate the gut in Schneider's Drosophila medium at RT before tissue staining is performed. Dissolve 1 µL of DHE dye (from the stock concentration of 30 mM) in 1 mL of Schneider's medium, making a final working concentration of 10-30 µM DHE dye.
3. Incubate the gut for 5 min at RT in dark, and then wash three times using Schneider's medium for every 5 min.
4. Fix the gut with 4% PF (optional step) and mount the gut onto glass slides, with anti-fade mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Capture images under a confocal microscope.

5. Measuring Fluorescence Using ImageJ Software

1. Import the captured fluorescence images acquired using fluorescence microscopy or confocal laser scanning microscopy into the ImageJ software.
2. Click on the Analyze menu and select Set measurements.
3. Select the output measure such as area integrated intensity and mean grey value.
4. Click Measure.
5. Select a region without fluorescence to set the background.
6. Export the data into the Excel spreadsheet and determine the corrected total cell fluorescence (CTCF), using the calculation as shown below.
   CTCF = Integrated Density - (Area of selected cell X Mean fluorescence of background readings)
7. Construct a bar chart and perform statistical analysis.

Results

NP-exposed cells were processed with the cell staining reagent kit, followed by cell sorting using flow cytometry. ZnO NP-treated cells (bottom, right panel) exhibit a higher percentage of early (R3)/ late apoptotic cells (R6) than control cells (R5, bottom, left panel). Necrotic cell death is denoted by R4 (top, right panel) (Figure 2). The results of the FITC/Annexin V Assay on ZnO NP-treated MRC-5 fibroblasts are shown in Figure 2

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

List of materials used in this article

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
ImageJ 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