Cytotoxicity Assays with Zebrafish Cell Lines

Irisdoris Rodrigues de Souza; Tain&#225; Wilke Sivek; J&#250;lia Beatriz Vaz de Oliveira; Andrezza Di Pietro Micali Canavez; Nat&#225;lia de Albuquerque Vita; Desiree Cigaran Schuck; Isisdoris Rodrigues de Souza; Marta Margarete Cestari; Marcio Lorencini; Daniela Morais Leme

doi:10.3791/64860

JoVE Journal > Environment

Medio ambiente

Cytotoxicity Assays with Zebrafish Cell Lines

Published: January 06, 2023

doi:

10.3791/64860

Irisdoris Rodrigues de Souza¹, Tainá Wilke Sivek¹, Júlia Beatriz Vaz de Oliveira¹, Andrezza Di Pietro Micali Canavez², Natália de Albuquerque Vita², Desiree Cigaran Schuck², Isisdoris Rodrigues de Souza¹, Marta Margarete Cestari³, Marcio Lorencini², Daniela Morais Leme³

¹Graduate Program in Genetics, Department of Genetics,Federal University of Paraná (UFPR), ²Safety of Product Department,Grupo Boticário, ³Department of Genetics,Federal University of Paraná (UFPR)

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

Start with a T75 flask of ZFL or ZEM2S cells with 80% confluence, cultured in the respective complete medium at 28 °C without CO₂.
Remove the culture medium from the flask and wash the cells by adding 10 mL of 1x phosphate-buffered saline (PBS) (0.01 M). Add 3 mL of 1x trypsin (0.05% v/v; 0.5 mM trypsin-EDTA) to the culture flasks. Incubate at 28 °C for 3 min.
Gently tap the flask to release the cells, and then stop the trypsin digestion by adding 3 mL of complete culture medium to the flask.
Transfer the cell suspension to a 15 mL conical centrifuge tube and centrifuge at 100 × g for 5 min.
After centrifugation, carefully remove the supernatant, add 1 mL of complete medium for ZFL or ZEM2S cells, and resuspend the pellet using a micropipette.

2. Cell counting by trypan blue dye exclusion

Add 10 µL of the cell suspension and 10 µL of trypan blue dye to a microtube to count the cells and evaluate their viability. Mix the cell suspension and dye using a pipette.
Then, transfer 10 µL of this mixture (cell suspension + trypan blue) to a Neubauer chamber and count the cells in the four large squares (Quadrants Q) placed at the corners of the chamber, considering viable cells to be those that do not take up trypan blue. Determine the number of viable cells using equation (1):
(1)
Calculate the final cell number in the cell suspension by multiplying the cell number determined using equation (1) by two (the dilution factor due to the use of trypan blue).
NOTE: Alternatively, an automated cell counting system (e.g., a cytomer with cell counting and viability function) can be used.

3. Cell plating in 96-well plates

Calculate the cell suspension volume needed to obtain the number of cells required to perform the cytotoxicity assays. The number of viable cells for each cell line is indicated below:
1. Plate 60,000 viable ZEM2S cells per well; thus, for the entire plate, use six million cells in 20 mL of complete medium (200 µL/well, 96-well plate).
2. Plate 40,000 viable ZFL cells per well; thus, for the entire plate, use four million cells in 20 mL of complete medium (200 µL/well, 96-well plate).
After that, transfer the respective volume of the cell suspension to a reagent reservoir (sterile) and fill up with the complete culture medium for ZFL or ZEM2S to 20 mL. Using a multichannel pipette, mix the solution gently up and down.
NOTE: Take care not to form foam or bubbles.
Add 200 µL of the cell suspension to each well of a transparent polystyrene 96-well plate using the multichannel micropipette. Incubate the plates at 28 °C for 24 h.
NOTE: The plate must have at least three wells without cells for the blank control, and only complete media should be added to these wells. The edge effect (caused by higher evaporation in the edge wells) commonly occurs in 96-well plate assays and can affect the viability of the cells in the edge wells of the plate²⁸. This effect can be higher or lower depending on the 96-well plate brand and design²⁸. Although we did not notice any cell growth/viability disturbance for ZFL and ZEM2S in the edge wells, we suggest sealing the plate with parafilm or adhesive sealing foil to prevent this effect, or culturing the cells only in the 60 inside wells and filling the edge wells with PBS.

4. Exposure of cells to test chemical

Carefully discard the spent media from the wells using a multichannel micropipette.
Expose the cells to test chemicals at different concentrations. Prepare the solutions of the test chemical concentrations in the culture media for ZFL or ZEM2S without fetal bovine serum (FBS) (exposure media). Then, add 100 µL per well of these solutions in technical triplicate (i.e., three wells/test chemical concentration).
For controls, place the control groups on the same plate as the test chemical in technical triplicates (three wells/control group). Thus, for the blank control (B), add 100 µL of the exposure media in the cell-free wells, for the negative control (NC), add 100 µL of the exposure media to wells with cells, and for the positive control (PC), expose the cells to a solution of 1% Triton X-100 prepared in the exposure media. In some cases, a solvent control (SC) should be included in the plate, considering a clearly non-cytotoxic concentration as a final solvent concentration.
NOTE: It is recommended to use 0.5% DMSO as solvent; DMSO can be used up to 1% as solvent in these cell lines without exceeding the cytotoxicity threshold of 10% related to the negative control.
Incubate the plates at 28 °C for 24 h. Seal the plates with parafilm or adhesive sealing foil to prevent culture medium evaporation.
NOTE: Certain chemicals may have intrinsic background absorbance or fluorescence that may interfere with the absorbance or fluorescence of the indicator dye(s) (e.g., compounds with color may influence absorbance, serum albumin²⁹, and compounds interfering with reduction enzymes³⁰^,³¹). In this case, the plate must include an additional control by adding test chemical solutions in the wells without cells. This is to verify the possible interference of the chemical auto-absorbance/autofluorescence with the dyes. If interference is detected, one should evaluate whether it can be excluded to obtain a correct prediction of cytotoxicity.

5. Cytotoxicity assays

NOTE: Prepare all solutions according to Table 1. All the steps described below (Figure 1) are carried out under sterile conditions. The use of a pipette to discard the exposure media is not recommended, because the cells can easily detach from the wells after chemical treatment.

AB and CFDA-AM assays
1. After 24 h of test chemical exposure, carefully discard the exposure media by pouring the content into a collection tray.
2. Wash the plate with 200 µL of PBS. Carefully remove the PBS by pouring it into a collection tray to avoid losing cells.
3. Add 100 µL per well of AB/CFDA-AM solution. Incubate the plate for 30 min in the dark at 28 °C.
4. Measure the fluorescence in a fluorescence plate reader at 530 nm (excitation) and 595 nm (emission) for AB, and at 493 nm (excitation) and 541 nm (emission) for CFDA-AM.
NR assay
NOTE: The steps for the NR assay are carried out immediately after the AB and CFDA-AM assays (Figure 1).
1. Centrifuge the NR working solution (40 µg/mL) at 600 × g for 10 min.
  NOTE: Precipitation of NR in the tube must not be transferred to the plates. Thus, after centrifugation of the NR working solution, collect the supernatant using a pipette without aspirating the NR precipitates. Transfer the supernatant to a reagent reservoir.
2. Carefully remove the AB/CFDA-AM solution by pouring the content into a collection tray.
3. Add 100 µL per well of the NR working solution using a multichannel micropipette. Incubate the plate at 28 °C for 3 h.
  NOTE: After the 3 h incubation, observe if NR precipitation occurred in the plates using a microscope. NR precipitates may interfere with the quantification of the cell viability, thus, they should not be present.
4. Carefully remove the NR solution by pouring the content into a collection tray. Wash the wells by adding 150 µL of PBS per well.
5. Add 150 µL per well of the NR extraction solution and incubate the plate on a plate shaker for 10 min for gently shaking. Measure the absorbance at 540 nm in a plate reader.
  NOTE: A second readout at 690 nm should be carried out to exclude any background fingerprint absorbance in the plate.
MTT assay
NOTE: The MTT assay must be carried out separately from the assays described above (in a new plate) (Figure 2).
1. Carefully remove the exposure media by pouring the content into a collection tray.
2. Add 100 µL of MTT working solution per well using a multichannel micropipette. Incubate the plate at 28 °C for 4 h.
3. Discard the MTT solution by pouring the content into a collection tray.
4. Add 100 µL per well of DMSO to extract the formazan crystals, incubating the plate on a plate shaker for 10 min. Measure the absorbance at 570 nm using a plate reader.
  NOTE: A second readout at 690 nm should be carried out to exclude any background fingerprint absorbance in the plate. It is important to note that test chemicals may interfere with MTT, which must be evaluated to ensure the quality of the generated data³². For this, cell-free wells containing the test concentrations and MTT (0.5 mg/mL) should be exposed, followed by incubation to observe any color change in the wells that may increase the absorbance and lead to false viability results. Chemicals that interact with MTT must be avoided in this test.

6. Calculating cell viability/cytotoxicity

NOTE: The raw absorbance or fluorescence acquired is used to calculate cell viability as a percentage related to the negative control (for test chemicals prepared directly in exposure media) or solvent control (for test chemicals prepared using solvents, such as DMSO). Before determining the cell viability percentage, the raw data must be normalized by the blank control.

Calculate the average absorbance or fluorescence for each test chemical concentration and control group (three wells/treatment).
To determine the cell viability percentage relative to control (negative or solvent), use equation (2):
(2)
NOTE: Absorbance (abs) or fluorescence (fluo) units represent the mean of absorbance or fluorescence measured in the three wells per concentration; blank represents wells without cells.

Representative Results

Figure 3 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 the color of the wells on the plate; however, the fluorescence is higher in wells containing viable cells due to the retention of CFDA-AM and subsequent conversion into carboxyfluorescein (fluorescent substance).

For the NR assay (Figure 3B), the blank wells must be transparent with very low absorbance values since there are no cells to retain the NR dye. In some cases, the blank wells are not transparent, indicating the occurrence of NR precipitation on the plate; in this instance, this should not be considered a valid experiment. Highly cytotoxic concentrations of the test chemicals and PC are transparent or present a very light pink color with low absorbance values, while wells containing viable cells retain the NR dye and present a dark pink color and high absorbance values.

For the MTT assay (Figure 3C), the blank wells must be transparent and with very low absorbance as there are no cells to convert MTT into formazan. Highly cytotoxic concentrations of the test chemicals and PC are transparent or present a very light violet color with low absorbance values, while wells containing viable cells transform the MTT (yellow) into formazan (violet substance), presenting a darker violet color with high absorbance values.

Figure 4A shows a representative graphic of cell viability after the calculation, using the averages of fluorescence or absorbance values per group. The graphic can be plotted with the input of viability percentage values, calculated by the viability calculation formula presented in protocol section 6, using data analysis software. The viability of cells exposed to the SC should not be 10% lower than in the NC¹⁷. The cell viability percentage for the test chemicals is calculated related to the NC or SC, depending on their solubility. In this case, different concentrations of DMSO are used as a test substance and the cell viability is related to NC, which is defined as 100% viability.

The cell viability data can be used to calculate the test chemicals' half maximal inhibitory concentration (IC₅₀) by logarithmic transformation, and interpolated standard curve by nonlinear regression after appropriate replicates³³^,³⁴^,³⁵. Figure 4B shows the IC₅₀, calculated from the viability percentage shown in Figure 4A. The IC₅₀ was obtained with at least three technical replicates and three experimental replicates using nominal concentrations In the AB assay. The analyses were performed with five different concentrations of the test substances; however, a higher number of concentrations may be required depending on the type of experiment. For instance, eight test concentrations are recommended for the range-finding test, which is usually performed to determine final test concentrations of a chemical substance for an experiment. As the viability assays have different cytotoxicity endpoints, we recommend calculating the IC₅₀ for each assay performed separately to identify differences in sensitivity caused by different mechanisms of action of the chemical substances or differences in sensitivity among cell lines. The differences in IC₅₀ values of tested chemicals in different cell lines may also vary depending on the type of culture medium used, since differences in composition related to proteins and lipids can impact the chemical bioavailability³⁶. In addition, the cytotoxicity of many chemicals can be evaluated through these assays. The IC₅₀ of other chemicals evaluated in ZFL and ZEM2S cell lines is shown in Figure 4B–H.

Figure 1: Schematical protocol of the AB, CFDA-AM, and NR assays performed in the same 96-well plate. Abbreviations: AB = Alamar Blue; CFDA-AM = 5-carboxyfluorescein diacetate acetoxymethyl ester; NR = Neutral Red; PBS = phosphate-buffered saline. Please click here to view a larger version of this figure.

Figure 2: Schematical protocol of the MTT assay performed in a 96-well plate. Abbreviation: MTT = 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide. Please click here to view a larger version of this figure.

Figure 3: Representative results of the AB, CFDA-AM, NR, and MTT assays. The images show the color differences in the wells for controls and test concentrations in (A) AB and CFDA-AM, (B) NR, and (C) MTT assays. Abbreviations: AB = Alamar Blue; CFDA-AM = 5-carboxyfluorescein diacetate acetoxymethyl ester; NR = Neutral Red; PBS = phosphate-buffered saline; B = blank wells (cell-free wells); SC = solvent control (0.5% DMSO); NC = negative control (cells in culture medium); PC = positive control (1% Triton X-100). Please click here to view a larger version of this figure.

Figure 4: Calculation of cell viability and viability data for different test chemicals. The calculation of cell viability using the readout data can be expressed as the percentage (%) of cell viability related to the NC or SC (A). The cytotoxicity of a chemical can be assessed by using the viability data to interpolate a standard curve by nonlinear regression for different test chemicals (B–H). Data is represented as mean of cell viability (dots) and standard deviation (bars) of three technical replicates and three experimental replicates (AB assay). Please click here to view a larger version of this figure.

Figure 5: MTT assay performed in ZFL and ZEM2S cells cultured in the presence (10%) and absence (0%, completely FBS-deprived) of FBS for 24 h. (A) ZFL cells at 0% and 10% FBS show no significant difference in cell viability by Kruskall-Wallis test (p = 0.2286). (B) ZEM2S cells at 0% and 10% FBS show no significant difference in cell viability by Mann-Whitney test (p = 0.3429). A significance level of p < 0.05 was considered. Data is expressed as median and interquartile range of three technical replicates. Abbreviations: n.s. = no significant difference; FBS = fetal bovine serum; MTT = 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide. Please click here to view a larger version of this figure.

Figure 6: MTT and NR assays performed in ZFL and ZEM2S cells treated (24 h) with DMSO at different concentrations (0.1%, 0.5%, and 1%) and negative control. DMSO-treated ZFL cells at any tested concentration did not show a significant difference in cell viability related to NC by the (A) MTT assay (p = 0.074) and (B) NR assay (p = 0.216). DMSO-treated ZEM2S cells did not show a significant difference in cell viability related to NC by the (C) MTT assay (p = 0.422) and (D) NR assay (p = 0.287). A significance level of p < 0.05 in the Kruskall-Wallis test was considered. Data is expressed as median and interquartile range of three technical replicates. Abbreviations: n.s. = no significant difference; NC = negative control; MTT = 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; NR = Neutral Red. Please click here to view a larger version of this figure.

Table 1: Solutions and media used in this protocol. Please click here to download this Table.

Discussion

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 integrity), the combination thereof provides an accurate evaluation of chemical cytotoxicity in zebrafish cell lines. This protocol also recommends the culture of ZFL and ZEM2S cell lines in a CO₂-free condition, due to their culture media composition that can adequately buffer the culture system, maintaining pH at 7.4 (physiological pH). The culture media composition and the CO₂-free environment proposed in this protocol for both cell lines are widely reported in the literature. The ZFL cell line is usually cultured in L-15 and RPMI media with or without the addition of sodium bicarbonate and without CO₂³⁷^,³⁸^,³⁹^,⁴⁰^,⁴¹^,⁴²^,⁴³. Meanwhile, the ZEM2S cell line is cultured according to instructions of the bioresource center, and its culture media is formulated for CO₂-free cultures; thus, CO₂ and air mixture can be detrimental to cells when using this type of culture media⁴⁴.

The chemical exposure in a culture medium without adding FBS was performed based on published studies, showing that the chemical substances' bioavailability in in vitro assays is significantly impacted by their binding to serum proteins. For instance, Chen et al.⁴⁵ showed that the presence of serum proteins in the RTgill-W1 assay could reduce the bioavailability of a cationic surfactant (C12-benzalkonium) up to three-and-a-half-fold. The chemical bound to FBS generally ranged from 47% to 90% in the culture medium⁴⁵. Thus, to avoid this issue, we evaluated the cell viability of ZFL and ZEM2S cells in cultures completely deprived of FBS for 24 h using the MTT assay. The results showed no significant difference in cell viability from ZFL or ZEM2S cultures with (10%) and without (0%) FBS, indicating that these zebrafish cell lines can be subject to chemical treatment in culture media deprived of FBS (Figure 5). It is important to highlight that decreasing the amount of FBS might cause other consequences in chemical bioavailability. For instance, lipophilic chemicals have higher sorption to plastic labware and plates, reducing chemical bioavailability³⁶. However, the presence of FBS in culture medium can decrease plastic binding due to serum constituents competing with the plastic for binding to the chemicals⁴⁶. The best decision related to the percentage of FBS supplementation or its complete deprivation might depend on the type of the test chemical. For example, Pomponio et al.⁴⁶ reported that although the chemical amiodarone has a higher binding to plastic in the absence of FBS, its bioavailability is even lower when using 10% FBS. For mono-N-desethylamiodarone, almost the same amount is bound to the serum medium and to the walls⁴⁶.

Organic solvents (e.g., DMSO) are generally recommended to be used up to 0.5% in in vitro assays. However, this low concentration can impair testing higher concentrations of poor water-soluble chemicals. To prevent this issue, we evaluated whether higher concentrations of DMSO were suitable for ZFL and ZEM2S cell lines not exceeding the cytotoxicity threshold of 10%. For this, non-treated cells (NCs) and cells treated (24 h) with different concentrations of DMSO (0.1%, 0.5%, and 1%) were processed for the MTT and NR assays. The results showed no significant difference in cell viability from the treatment group compared to NCs (Figure 6), indicating that, under these particular conditions, concentrations of DMSO up to 1% can be used. Other fish cell lines also support DMSO concentrations higher than 0.5%, which is not a particularity of ZFL and ZEM2S cell lines. For instance, maximal solvent concentrations of up to 2% DMSO (with no cytotoxic effect observed) have been applied in cytotoxicity assays using CHSE-214 (cell line derived from Oncorhynchus tshawytscha embryo)⁴⁷, RTG-2 (Oncorhynchus mykiss gonadal cell line)⁴⁸^,⁴⁹^,⁵⁰, and PLHC-1 (Poeciliopsis lucida hepatocellular carcinoma cell line)⁴⁸ cells. The maximum solvent concentration of 1% DMSO has also been used in cytotoxicity assays using RTL-W1 (Oncorhynchus mykiss gonadal cell line)⁵¹ and CCO (Ictalurus punctatus ovary cell line)⁵² cells. According to Mori and Wakabayashi⁴⁷, fish cell lines may have a lower sensitivity to DMSO than mammalian cell lines. However, it is important to highlight that the maximal acceptable concentration of 1% DMSO was exclusively defined for cytotoxicity, and this should be carefully evaluated for other endpoints (e.g., genotoxicity, epigenetics, protein-coding gene expression analysis) in ZFL and ZEM2S cell lines.

The MTT and AB assays are based on metabolic activity to determine cell viability. Although the MTT assay is the most used viability assay, in comparison to the AB assay it can be slightly less sensitive, overestimating cell viability in some cases⁵³. The higher sensitivity of the AB assay may be related to the measurement method, as fluorescence measurement is more sensitive than colorimetric measurement¹⁵. Nonetheless, both the AB and MTT assays are high-quality assays to identify cytotoxic chemical substances, and their generated data have been used to classify hazardous chemical substances according to their intrinsic cytotoxic potential⁵³.

The idea of performing the AB, CFDA-AM, and NR assay in the same plate was based on the OECD TG 249 (RTgill-W1 assay)¹⁷; however, modifications were made to perform these assays in 96-well plates, as well as to be suitable for zebrafish cell lines. Assays in 96-well plates, instead of 24-well plates, can be advantageous for high-throughput cytotoxicity testing in fish cell lines. In addition, the RTgill-W1 assay uses only L-15 culture medium, while the ZFL and ZEM2S cell lines are cultured in a medium containing D-glucose. The culture medium makes it possible to perform the MTT assay in these cell lines, as cell lines cultured in glucose-free medium (e.g., only L-15) may immediately decrease the MTT reduction in cells and impair the performance of this assay⁵⁴. This protocol allows four different viability assays to be easily performed in two zebrafish cell lines.

This protocol can be used to study the effects of chemical substances on fish by using in vitro models. Different cell lines may have different sensitivities to estimate chemical effects, even though they are from the same group of vertebrates, such as fish. For instance, comparing ZFL and ZF4 cell lines with fish embryos, Langu-Mitea et al.²¹ demonstrated that ZFL is capable of generating similar results compared to the fish embryo toxicity test. Tanneberger et al.⁵ showed that the permanent fish cell lines GSF, PLHC, RTG-2, RTgill-W1, and R1 have different sensitivities in predicting chemical fish toxicity compared to in vivo (adult fish). Although RTgill-W1 (permanent fish gill cell line) was recently validated as an alternative method to predict fish acute toxicity, the applicability of other cell lines in ecotoxicity studies should be investigated. Using cell lines from different fish species and tissue origins as well as developmental stages (ZEM2S: embryo; ZFL: adult liver) may significantly contribute to ecotoxicity studies, since it can address effects related to specific stages of fish development and target organs. Thus, chemical effects should be investigated using different cell cultures reﬂecting different target sites in ﬁsh (e.g., liver, gonads, gill, brain), and not only in a single cell line⁵⁵.

These protocols can have different applications in ecotoxicity studies, and their use is not necessarily limited to predicting fish acute toxicity. For instance, they can be applied to define sub-cytotoxic concentrations to perform other in vitro fish toxicity testing, such as evaluating endocrine-disrupting effects on fish. In addition, the in vitro cytotoxicity data can also help develop and improve physiologically-based toxicokinetic (PBTK) modeling. As PBTK focuses on the distribution of a chemical in an organism considering the different organs (such as the gill, liver, or intestine)⁵⁶, using cell lines from different fish species and tissue origins can be a useful source of information for this model. The results of in vitro bioassays (e.g., cytotoxicity assays) provide input data representing biological processes in the PBTK model and contribute to in vitro a in vivo extrapolation²¹^,⁵⁶.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

In memory of Dr. Márcio Lorencini, a coauthor of this work, an excellent researcher in the ﬁeld 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 ﬁnancial support of the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) (Finance Code 001) and the Grupo Boticario.

Materials

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

Referencias

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Rodrigues de Souza, I., Wilke Sivek, T., Vaz de Oliveira, J. B., Di Pietro Micali Canavez, A., de Albuquerque Vita, N., Cigaran Schuck, D., Rodrigues de Souza, I., Cestari, M. M., Lorencini, M., Leme, D. M. Cytotoxicity Assays with Zebrafish Cell Lines. J. Vis. Exp. (191), e64860, doi:10.3791/64860 (2023).