Name: Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster (Video) | JoVE Journal
Uploaded: 2019-09-19T13:30:00.000+00:00
Duration: 6 min 47 s
Description: Learn nanotoxicity assessment using zinc oxide nanoparticles in human MRC5 lung fibroblasts and Drosophila melanogaster. This protocol measures cell death types and reactive oxygen species production via flow cytometry and imaging.

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5 minutes

The study was supported by the grant number R706-000-043-490. The study does not represent the view of the grant sponsor.

1. Fluorescence Activated Cell Sorting (FACS) Analysis on Lived/Fixed cells
<ol>
	<li>Sonicate ZnO NPs in suspension for 15 min.</li>
	<li>Prepare ZnO NPs at various concentrations (e.g., 0, 10, 25, 50,100 and 200 &#181;g/mL) using 1 mg/mL ZnO NP stock solution for the treatment of cultured cells.</li>
	<li>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.</li>
	<li>At each time point, collect the cells by centrifuging at 300 x g for 5 min.</li>
	<li>Wash the cell pellets twice with phosphate buffered saline (PBS).</li>
	<li>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 &#181;L.</li>
	<li>Add 5 &#181;L of Fluorescein isothiocyanate (FITC) Annexin V stain and 5 &#181;L of propidium iodide (PI) DNA stain, and incubate the cells for 15 min at room temperature (RT; 25 &#176;C) in dark.</li>
	<li>Top up the samples with an additional 400 &#181;L of 1x binding buffer before sorting the cells by flow cytometry. A minimum of 10,000 cells is analyzed for each sample.</li>
	<li>Tabulate the bar chart using the median intensity obtained.</li>
</ol>
2. Exposure of ZnO NPs to Drosophila
<ol>
	<li>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.</li>
	<li>Mix the nanoparticles with food thoroughly in the vials using the pipette.</li>
	<li>Allow fly food containing ZnO NPs to cool for at least 2-3 h before use.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
3. Dissection of Fly
<ol>
	<li>Collect late 3rd instar larvae from the wall of the vials for analyses. Freshly laid eggs normally develop into late 3rd instar larvae after 72-120 h at RT.</li>
	<li>Clean the dissection dish and fill up the well with dissection medium/PBS.</li>
	<li>Dissect the larvae (late 3rd instar) under the stereomicroscope, using a pair of forceps.</li>
	<li>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&#39;s Drosophila medium, prior to the fixing step using 1 mL of 4% paraformaldehyde (PF).</li>
	<li>3.5Fix the gut in PF for 10 min at RT, for subsequent experiments, such as immunostaining.</li>
</ol>
4. ROS Detection Using Dihydroethidium (DHE) Staining
<ol>
	<li>Treat larvae with various concentrations of ZnO NPs as described in step 2.1.</li>
	<li>Following the dissection of the gut from 3rd instar larvae as described under section 3, incubate the gut in Schneider&#39;s Drosophila medium at RT before tissue staining is performed. Dissolve 1 &#181;L of DHE dye (from the stock concentration of 30 mM) in 1 mL of Schneider&#39;s medium, making a final working concentration of 10-30 &#181;M DHE dye.</li>
	<li>Incubate the gut for 5 min at RT in dark, and then wash three times using Schneider&#39;s medium for every 5 min.</li>
	<li>Fix the gut with 4% PF (optional step) and mount the gut onto glass slides, with anti-fade mounting medium containing 4&#8242;,6-diamidino-2-phenylindole (DAPI). Capture images under a confocal microscope.</li>
</ol>
5. Measuring Fluorescence Using ImageJ Software
<ol>
	<li>Import the captured fluorescence images acquired using fluorescence microscopy or confocal laser scanning microscopy into the ImageJ software.</li>
	<li>Click on the Analyze menu and select Set measurements.</li>
	<li>Select the output measure such as area integrated intensity and mean grey value.</li>
	<li>Click Measure.</li>
	<li>Select a region without fluorescence to set the background.</li>
	<li>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)</li>
	<li>Construct a bar chart and perform statistical analysis.</li>
</ol>

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The authors declare that they have no competing financial interests.

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...

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 applications1,2,3.
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 genotoxicity4,5,6,7,8. 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 transportation4,7,9,10. 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, zebrafish11,12, 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 questions17,18,19,20,21. In recent years, a number of studies related to immunotoxicity using different types of NPs in Drosophila have been reported15,22,23,24. 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.

<table><tbody><tr><td>15% Methyl 4-Hydroxybenzoate</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>4% Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>Bacto Agar</td><td>BD biosciences</td><td></td><td></td></tr><tr><td>&lt;em&gt;cncCK6/TM3, Sb&lt;/em&gt;</td><td></td><td></td><td>a gift from Dr. Kerppola T</td></tr><tr><td>Cornmeal, glucose, yeast brewer</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>CyAn ADP with Summit Software</td><td>DAKO</td><td></td><td>https://flow.usc.edu/files/2014/07/BC-Cyan-ADP-User-Guide-2016.pdf</td></tr><tr><td>Dihydroethidium (Hydroethidine)</td><td>Thermo Fisher Scientific</td><td>D11347</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD biosciences</td><td>556547</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Glucolin</td><td>Supermarket</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td></td></tr><tr><td>MRC5 human lung fibroblast</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; medium</td><td>Thermo Fisher Scientific</td><td>21720-024</td><td></td></tr><tr><td>Vectashield antifade mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td></td></tr><tr><td>Wild-type &lt;em&gt;Canton-S; Sod2N308/CyO&lt;/em&gt;</td><td>NIG-FLY</td><td></td><td></td></tr><tr><td>Zinc Oxide Nanoparticles</td><td>Sigma Aldrich</td><td>721077</td><td>Refer Sheet 2</td></tr></tbody></table>

toxicity study of zinc oxide nanoparticles in cell culture and in drosophila melanogaster

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

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.

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</str...

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

In this article, zebrafish embryos are used for chemical toxicity screening. Chemical toxicity is correlated with phenotype readouts. The sample protocol shows a methodological approach of performing the chemical toxicity screening.

Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes.  Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Methods to Test Endocrine Disruption in Drosophila melanogaster

This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Cytotoxicity Assays with Zebrafish Cell Lines

This protocol describes an efficient and inexpensive method that uses liquid media to assess the effects of chemical toxicants on the viability of adult Drosophila melanogaster.

Assessment of Chemical Toxicity in Adult Drosophila Melanogaster

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

The protocol presents a high-throughput, cost-effective method for assessing neurotoxicity in the fruit fly through the quantification of locomotor dysfunction as an alternative to traditional mammalian models. It aims to evaluate the neurotoxic effects of pharmaceutical compounds, environmental agents, or genetic modifications using a sensitive, reproducible, and ethically favorable model.

Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

We present a protocol to produce antigen-specific mouse T-cells using retroviral...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Learn nanotoxicity assessment using zinc oxide nanoparticles in human MRC5 lung fibroblasts and Drosophila melanogaster. This protocol measures cell death types and reactive oxygen species production via flow cytometry and imaging.

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

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