Name: toxicity study of zinc oxide nanoparticles in cell culture and in drosophila melanogaster
Uploaded: 2019-09-19T13:30:00
Description: Watch this Scientific Journal Video about Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster at JoVE.com

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 eac...

<ol>
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W., Leung, P. T., Djurisic, A. B., Leung, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicities+of+nano+zinc+oxide+to+five+marine+organisms:+influences+of+aggregate+size+and+ion+solubility.">Toxicities of nano zinc oxide to five marine organisms: influences of aggregate size and ion solubility.</a> Analytical and Bioanalytical Chemistry. 396, 609-618 (2010).</li><li>Hua, J., Vijver, M. G., Richardson, M. K., Ahmad, F., Peijnenburg, W. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophotoxicology:+the+growing+potential+for+Drosophila+in+neurotoxicology.">Drosophotoxicology: the growing potential for Drosophila in neurotoxicology.</a> Neurotoxicology and Teratology. 32, 74-83 (2010).</li><li>Ong, C., Yung, L. Y., Cai, Y., Bay, B. H., Baeg, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+organism+to+study+nanotoxicity.">Drosophila melanogaster as a model organism to study nanotoxicity.</a> Nanotoxicology. 9, 396-403 (2015).</li><li>Hoffmann, J. A., Reichhart, J. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+sequence+to+phenotype:+reverse+genetics+in+Drosophila+melanogaster.">From sequence to phenotype: reverse genetics in Drosophila melanogaster.</a> Nature Reviews Genetics. 3, 189-198 (2002).</li><li>Adams, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Drosophila+melanogaster.">The genome sequence of Drosophila melanogaster.</a> Science. 287, 2185-2195 (2000).</li><li>Ong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+nanoparticles+disrupt+germline+stem+cell+maintenance+in+the+Drosophila+testis.">Silver nanoparticles disrupt germline stem cell maintenance in the Drosophila testis.</a> Scientific Reports. 6, (2016).</li><li>Alaraby, M., Demir, E., Hernandez, A., Marcos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+potential+harmful+effects+of+CdSe+quantum+dots+by+using+Drosophila+melanogaster+as+in+vivo+model.">Assessing potential harmful effects of CdSe quantum dots by using Drosophila melanogaster as in vivo model.</a> Science of the Total Environment. 530-531, 66-75 (2015).</li><li>Barik, B. K., Mishra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+as+a+potential+teratogen:+a+lesson+learnt+from+fruit+fly.">Nanoparticles as a potential teratogen: a lesson learnt from fruit fly.</a> Nanotoxicology. , 1-27 (2018).</li><li>Jovanovic, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+a+human+food+additive,+titanium+dioxide+nanoparticles+E171,+on+Drosophila+melanogaster+-+a+20+generation+dietary+exposure+experiment.">The effects of a human food additive, titanium dioxide nanoparticles E171, on Drosophila melanogaster - a 20 generation dietary exposure experiment.</a> Scientific Reports. 8, (2018).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Toxicity+of+Nanoparticles+to+Human+Endothelial+Cells.">The Toxicity of Nanoparticles to Human Endothelial Cells.</a> Advances in Experimental Medicine and Biology. 1048, 59-69 (2018).</li><li>Akhtar, M. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>cncCK6/TM3, Sb</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>Image J 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&#8217;s Drosophila 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 Canton-S; Sod2N308/CyO</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

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...

Watch this Scientific Journal Video about Toxicity Study of Zinc Oxide Nanoparticles in Cell Culture and in Drosophila melanogaster at JoVE.com

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

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.

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 ...

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