Name: cytotoxicity assays with zebrafish cell lines
Uploaded: 2023-01-06T14:30:00
Description: Watch this Scientific Journal Video about Cytotoxicity Assays with Zebrafish Cell Lines at JoVE.com

In memory of Dr. M&#225;rcio Lorencini, a coauthor of this work, an excellent researcher in the &#64257;eld of cosmetics and devoted to promoting cosmetic research in Brazil. The authors are grateful for the Multi-user Laboratory in the Physiology Department (UFPR) for equipment availability and for the &#64257;nancial support of the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) (Finance Code 001) and the Grupo Boticario.

NOTE: See the Table of Materials for the list of materials used in this protocol and Table 1 for the composition of solutions and media used in this protocol.
1. Preparing ZFL and ZEM2S cells
<ol>
	<li>Start with a T75 flask of ZFL or ZEM2S cells with 80% confluence, cultured in the respective complete medium at 28 &#176;C without CO2.</li>
	<li>Remove the culture medium from the flask and wash the cells by adding 10 mL of 1x phos...

Cytotoxicity assays are widely used for in vitro toxicity evaluation, and this protocol article presents four commonly used cytotoxicity assays modified to be performed in zebrafish cell lines (i.e., cell density for 96-well plate, incubation time in the MTT assay, FBS depletion during the chemical exposure condition, and maximal acceptable concentration for the SC). As these assays quantify cytotoxicity by different cell viability endpoints (metabolic function, lysosomal membrane integrity, and cell membrane in...

Chemical substances need to be tested regarding their safety for human health and the environment. Molecular and cellular biomarkers have been increasingly considered in safety assessments to predict effects on living organisms by regulatory agencies and/or legislations (e.g., REACH, OECD, US EPA)1,2, since they can precede the in vivo adverse outcome (e.g., endocrine disruption, immunological response, acute toxicity, phototoxicity)3,4,5,6,7. In this context, cytotoxicity has been taken as a measurement to predict fish acute toxicity5,8; however, it can have many other applications in ecotoxicity studies, such as defining sub-cytotoxic concentrations of chemical substances to study their most diverse set of effects on fish (e.g., endocrine-disrupting effects).
In cell culture systems (in vitro systems), the cytotoxicity of chemical substances can be determined by methods differing in the types of endpoints. For instance, a cytotoxicity method can be based on an endpoint related to specific morphology observed during the cell death process, while another can determine cytotoxicity by the measurement of cell death, viability and functionality, morphology, energy metabolism, and cell attachment and proliferation. Chemical substances can affect cell viability through different mechanisms, thus cytotoxicity assessment covering different cell viability endpoints is necessary to predict chemical effects9.
MTT and Alamar Blue (AB) are assays that determine effects on cell viability based on cell metabolic activity. The MTT assay evaluates the activity of the mitochondrial enzyme succinate dehydrogenase10. The reduction of yellowish 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan blue occurs only in viable cells, and its optical density is directly proportional to the number of viable cells10. The AB assay is a sensitive oxidation-reduction indicator, mediated by mitochondrial enzymes that fluoresce and change color upon reducing resazurin to resorufin by living cells11; however, cytosolic and microsomal enzymes also contribute to the reduction of AB and MTT12. These enzymes may include several reductases, such as alcohol and aldehyde oxidoreductases, NAD(P)H: quinone oxidoreductase, flavin reductase, NADH dehydrogenase, and cytochromes11.
The Neutral Red (NR) assay is a cell viability assay based on the incorporation of this dye into the lysosomes of viable cells13. The uptake of NR depends on the capacity of the cells to maintain pH gradients. The proton gradient inside the lysosomes maintains a pH lower than the cytoplasm. At normal physiological pH, the NR presents a net charge of approximately zero, which enables it to penetrate cell membranes. Thus, the dye becomes charged and is retained inside the lysosomes. Consequently, the greater the amount of retained NR, the greater the number of viable cells14. Chemical substances that damage the cell surface or lysosomal membranes impair the uptake of this dye.
The CFDA-AM assay is a fluorometric cell viability assay based on the retention of 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)15. 5-CFDA-AM, an esterase substrate, is converted into carboxyfluorescein, a fluorescent substance that is polar and nonpermeable by membranes of living cells15; thus, it is retained in the inner side of an intact cell membrane, indicating viable cells.
Recently, three cytotoxicity assays (CFDA-AM, NR, and AB assays) were combined in a validated ISO (International Organization for Standardization) guideline (ISO 21115:2019)16 and OECD (Organization for Economic Co-operation and Development) test method (OECD TG 249) to evaluate fish acute toxicity using the RTgill-W1 cell line (permanent cell line from rainbow trout [Oncorhynchus mykiss] gill) in 24-well plates17. Although there is an existing cell-based method to predict fish acute toxicity, efforts have been invested in developing similar methods with other fish species and increasing the throughput of the method. Some examples include the development of ZFL cell lines transfected with reporter genes for specific toxicity pathways18,19, phototoxicity tests in the RTgill-W1 cell line20, and the use of ZFL and ZF4 cell lines (zebrafish fibroblastic derived from 1-day-old embryos) to assess toxicity by several cytotoxicity assays21.
Danio rerio (zebrafish) is one of the main fish species used in aquatic toxicity studies; thus, cell-based methods with zebrafish cell lines for fish toxicity testing may be extremely useful. The ZFL cell line is a zebrafish epithelial hepatocyte cell line that presents the main characteristics of liver parenchymal cells and can metabolize xenobiotics7,22,23,24,25. Meanwhile, the ZEM2S cell line is an embryonic zebrafish fibroblastic cell line derived from the blastula stage that can be used to investigate developmental effects on fish26,27. Thus, this protocol describes four cytotoxicity assays (MTT, AB, NR, and CFDA-AM assays), with modifications to be performed with ZFL and ZEM2S cell lines in 96-well plates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-CFDA, AM (5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester)</td>
			<td>Invitrogen</td>
			<td>C1345</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plate, 96 well plate</td>
			<td>Sarstedt</td>
			<td>83.3924</td>
			<td>Surface: Standard, flat base</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>Powder, high glucose, pyruvate</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix, powder</td>
			<td>Gibco</td>
			<td>21700026</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco</td>
			<td>41300021</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Neutral red&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>N4638</td>
			<td>Powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Orbital shaker&#160;</td>
			<td>Warmnest</td>
			<td>KLD-350-BI</td>
			<td>22 mm rotation diameter</td>
		</tr>
		<tr>
			<td>Dulbeccos PBS (10X) with calcium and magnesium</td>
			<td>Invitrogen</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td>Powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>31800-014</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>SFB - Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Powder,&#160; bioreagent for molecular biology</td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide&#160; 98%</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEM2S cell line</td>
			<td>ATCC</td>
			<td>CRL-2147</td>
			<td>This cell line was kindly donated by Professor Dr. Michael J. 
			Carvan (University of Wisconsin, Milwaukee, USA)</td>
		</tr>
		<tr>
			<td>ZFL cell line</td>
			<td>BCRJ</td>
			<td>256</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxicity assays with zebrafish cell lines

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Figure 3&#160;shows the plates of the AB, CFDA-AM, NR, and MTT assays. For the AB assay (Figure 3A), the blank wells and wells with no or a reduced number of viable cells show blue color and low fluorescence, while the wells with a high number of viable cells are pinkish and present high fluorescence values due to the transformation of resazurin (AB) into resorufin (pinkish substance) by the viable cells. For the CFDA-AM assay, there is no visible difference in ...

Watch this Scientific Journal Video about Cytotoxicity Assays with Zebrafish Cell Lines at JoVE.com

Cytotoxicity Assays with Zebrafish Cell Lines

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.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute ...

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