Jove Protocol 64860

Cytotoxicity Assays with Zebrafish Cell Lines
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
1 2
Graduate Program in Genetics, Department of Genetics, Federal University of Paraná (UFPR) Safety of Product Department, Grupo
3
Boticário Department of Genetics, Federal University of Paraná (UFPR)
Corresponding Author
Abstract
Daniela Morais Leme
[email protected]
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
Citation
protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish
Rodrigues de Souza, I., Wilke Sivek, T., (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The
Vaz de Oliveira, J.B., Di Pietro Micali
Canavez, A., de Albuquerque Vita, N., cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and
Cigaran Schuck, D., Rodrigues de
MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and
Souza, I., Cestari, M.M., Lorencini, M.,
Leme, D.M. Cytotoxicity Assays with lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test
Zebrafish Cell Lines. J. Vis. Exp. (191),
substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and
e64860, doi:10.3791/64860 (2023).
CFDA-AM are carried out simultaneously, followed by NR on the same plate, while
Date Published the MTT assay is performed on a separate plate. The readouts for these assays are
January 6, 2023 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
DOI
toxicity of chemical substances on fish.
10.3791/64860
URL
jove.com/video/64860
Introduction
Chemical substances need to be tested regarding their context, cytotoxicity has been taken as a measurement to
safety for human health and the environment. Molecular and predict fish acute toxicity5 , 8 ; however, it can have many
cellular biomarkers have been increasingly considered in other applications in ecotoxicity studies, such as defining sub-
safety assessments to predict effects on living organisms cytotoxic concentrations of chemical substances to study their
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
Copyright © 2023 JoVE Journal of Visualized Experiments jove.com January 2023 • 191 • e64860 • Page 1 of 19
most diverse set of effects on fish (e.g., endocrine-disrupting the lysosomes maintains a pH lower than the cytoplasm.
effects). At normal physiological pH, the NR presents a net charge
of approximately zero, which enables it to penetrate cell
In cell culture systems (in vitro systems), the cytotoxicity of
membranes. Thus, the dye becomes charged and is retained
chemical substances can be determined by methods differing
inside the lysosomes. Consequently, the greater the amount
in the types of endpoints. For instance, a cytotoxicity method
of retained NR, the greater the number of viable cells14 .
can be based on an endpoint related to specific morphology
Chemical substances that damage the cell surface or
observed during the cell death process, while another can
lysosomal membranes impair the uptake of this dye.
determine cytotoxicity by the measurement of cell death,
viability and functionality, morphology, energy metabolism, The CFDA-AM assay is a fluorometric cell viability assay
and cell attachment and proliferation. Chemical substances based on the retention of 5-carboxyfluorescein diacetate
can affect cell viability through different mechanisms, acetoxymethyl ester (CFDA-AM)15 . 5-CFDA-AM, an esterase
thus cytotoxicity assessment covering different cell viability substrate, is converted into carboxyfluorescein, a fluorescent
endpoints is necessary to predict chemical effects9 . substance that is polar and nonpermeable by membranes of
living cells15 ; thus, it is retained in the inner side of an intact
MTT and Alamar Blue (AB) are assays that determine
cell membrane, indicating viable cells.
effects on cell viability based on cell metabolic activity.
The MTT assay evaluates the activity of the mitochondrial Recently, three cytotoxicity assays (CFDA-AM, NR,
enzyme succinate dehydrogenase10 . The reduction of and AB assays) were combined in a validated ISO
yellowish 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium (International Organization for Standardization) guideline
bromide (MTT) to formazan blue occurs only in viable cells, (ISO 21115:2019)16 and OECD (Organization for Economic
and its optical density is directly proportional to the number Co-operation and Development) test method (OECD TG
of viable cells10 . The AB assay is a sensitive oxidation- 249) to evaluate fish acute toxicity using the RTgill-W1 cell
reduction indicator, mediated by mitochondrial enzymes that line (permanent cell line from rainbow trout [Oncorhynchus
fluoresce and change color upon reducing resazurin to mykiss] gill) in 24-well plates17 . Although there is an
resorufin by living cells11 ; however, cytosolic and microsomal existing cell-based method to predict fish acute toxicity,
enzymes also contribute to the reduction of AB and MTT12 . efforts have been invested in developing similar methods
These enzymes may include several reductases, such as with other fish species and increasing the throughput of
alcohol and aldehyde oxidoreductases, NAD(P)H: quinone the method. Some examples include the development of
oxidoreductase, flavin reductase, NADH dehydrogenase, and ZFL cell lines transfected with reporter genes for specific
cytochromes11 . toxicity pathways18 , 19 , phototoxicity tests in the RTgill-W1
cell line20 , and the use of ZFL and ZF4 cell lines (zebrafish
The Neutral Red (NR) assay is a cell viability assay based
fibroblastic derived from 1-day-old embryos) to assess toxicity
on the incorporation of this dye into the lysosomes of viable
by several cytotoxicity assays21 .
cells13 . The uptake of NR depends on the capacity of the
cells to maintain pH gradients. The proton gradient inside
Danio rerio (zebrafish) is one of the main fish species 5. After centrifugation, carefully remove the supernatant,
used in aquatic toxicity studies; thus, cell-based methods add 1 mL of complete medium for ZFL or ZEM2S cells,
with zebrafish cell lines for fish toxicity testing may and resuspend the pellet using a micropipette.
be extremely useful. The ZFL cell line is a zebrafish
epithelial hepatocyte cell line that presents the main 2. Cell counting by trypan blue dye exclusion
characteristics of liver parenchymal cells and can metabolize

1. Add 10 µL of the cell suspension and 10 µL of trypan blue
xenobiotics7 , 22 , 23 , 24 , 25 . Meanwhile, the ZEM2S cell line is
dye to a microtube to count the cells and evaluate their
an embryonic zebrafish fibroblastic cell line derived from the
viability. Mix the cell suspension and dye using a pipette.
blastula stage that can be used to investigate developmental
2. Then, transfer 10 µL of this mixture (cell suspension +
effects on fish26 , 27 . Thus, this protocol describes four
trypan blue) to a Neubauer chamber and count the cells
cytotoxicity assays (MTT, AB, NR, and CFDA-AM assays),
in the four large squares (Quadrants Q) placed at the
with modifications to be performed with ZFL and ZEM2S cell
corners of the chamber, considering viable cells to be
lines in 96-well plates.
those that do not take up trypan blue. Determine the
Protocol number of viable cells using equation (1):
NOTE: See the Table of Materials for the list of materials (1)
used in this protocol and Table 1 for the composition of
3. Calculate the final cell number in the cell suspension by
solutions and media used in this protocol.
multiplying the cell number determined using equation (1)
by two (the dilution factor due to the use of trypan blue).
1. Preparing ZFL and ZEM2S cells
NOTE: Alternatively, an automated cell counting system
(e.g., a cytomer with cell counting and viability function)
1. Start with a T75 flask of ZFL or ZEM2S cells with 80%
can be used.
confluence, cultured in the respective complete medium
at 28 °C without CO2.
3. Cell plating in 96-well plates
2. Remove the culture medium from the flask and wash the
1. Calculate the cell suspension volume needed to obtain
cells by adding 10 mL of 1x phosphate-buffered saline
the number of cells required to perform the cytotoxicity
(PBS) (0.01 M). Add 3 mL of 1x trypsin (0.05% v/v; 0.5
assays. The number of viable cells for each cell line is
mM trypsin-EDTA) to the culture flasks. Incubate at 28
indicated below:
°C for 3 min.
1. Plate 60,000 viable ZEM2S cells per well; thus, for
3. Gently tap the flask to release the cells, and then stop
the entire plate, use six million cells in 20 mL of
the trypsin digestion by adding 3 mL of complete culture
complete medium (200 µL/well, 96-well plate).
medium to the flask.
4. Transfer the cell suspension to a 15 mL conical centrifuge

tube and centrifuge at 100 × g for 5 min.
without fetal bovine serum (FBS) (exposure media).
2. Plate 40,000 viable ZFL cells per well; thus, for
Then, add 100 µL per well of these solutions in technical
the entire plate, use four million cells in 20 mL of
triplicate (i.e., three wells/test chemical concentration).
complete medium (200 µL/well, 96-well plate).
3. For controls, place the control groups on the same plate
2. After that, transfer the respective volume of the cell
as the test chemical in technical triplicates (three wells/
suspension to a reagent reservoir (sterile) and fill up with
control group). Thus, for the blank control (B), add 100
the complete culture medium for ZFL or ZEM2S to 20 mL.
µL of the exposure media in the cell-free wells, for
Using a multichannel pipette, mix the solution gently up
the negative control (NC), add 100 µL of the exposure
and down.
media to wells with cells, and for the positive control
NOTE: Take care not to form foam or bubbles.
(PC), expose the cells to a solution of 1% Triton X-100
3. Add 200 µL of the cell suspension to each well
prepared in the exposure media. In some cases, a
of a transparent polystyrene 96-well plate using the
solvent control (SC) should be included in the plate,
multichannel micropipette. Incubate the plates at 28 °C
considering a clearly non-cytotoxic concentration as a
for 24 h.
final solvent concentration.
NOTE: The plate must have at least three wells without
NOTE: It is recommended to use 0.5% DMSO as solvent;
cells for the blank control, and only complete media
DMSO can be used up to 1% as solvent in these cell
should be added to these wells. The edge effect (caused
lines without exceeding the cytotoxicity threshold of 10%
by higher evaporation in the edge wells) commonly
related to the negative control.
occurs in 96-well plate assays and can affect the viability
4. Incubate the plates at 28 °C for 24 h. Seal the plates
of the cells in the edge wells of the plate28 . This effect
with parafilm or adhesive sealing foil to prevent culture
can be higher or lower depending on the 96-well plate
medium evaporation.
brand and design28 . Although we did not notice any cell
NOTE: Certain chemicals may have intrinsic background
growth/viability disturbance for ZFL and ZEM2S in the
absorbance or fluorescence that may interfere with
edge wells, we suggest sealing the plate with parafilm or
the absorbance or fluorescence of the indicator dye(s)
adhesive sealing foil to prevent this effect, or culturing the
(e.g., compounds with color may influence absorbance,
cells only in the 60 inside wells and filling the edge wells
serum albumin29 , and compounds interfering with
with PBS.
reduction enzymes30 , 31 ). In this case, the plate must
4. Exposure of cells to test chemical include an additional control by adding test chemical
solutions in the wells without cells. This is to verify the
1. Carefully discard the spent media from the wells using a
possible interference of the chemical auto-absorbance/
multichannel micropipette.
autofluorescence with the dyes. If interference is
2. Expose the cells to test chemicals at different
detected, one should evaluate whether it can be
concentrations. Prepare the solutions of the test chemical
excluded to obtain a correct prediction of cytotoxicity.
concentrations in the culture media for ZFL or ZEM2S
2. Carefully remove the AB/CFDA-AM solution by
5. Cytotoxicity assays
pouring the content into a collection tray.
NOTE: Prepare all solutions according to Table 1. All the
3. Add 100 µL per well of the NR working solution using
steps described below (Figure 1) are carried out under sterile
a multichannel micropipette. Incubate the plate at 28
conditions. The use of a pipette to discard the exposure media
°C for 3 h.
is not recommended, because the cells can easily detach
NOTE: After the 3 h incubation, observe if
from the wells after chemical treatment.
NR precipitation occurred in the plates using a
1. AB and CFDA-AM assays microscope. NR precipitates may interfere with the

quantification of the cell viability, thus, they should
1. After 24 h of test chemical exposure, carefully
not be present.
discard the exposure media by pouring the content
into a collection tray. 4. Carefully remove the NR solution by pouring the
content into a collection tray. Wash the wells by
2. Wash the plate with 200 µL of PBS. Carefully remove
adding 150 µL of PBS per well.
the PBS by pouring it into a collection tray to avoid
losing cells. 5. Add 150 µL per well of the NR extraction solution
and incubate the plate on a plate shaker for 10 min
3. Add 100 µL per well of AB/CFDA-AM solution.
for gently shaking. Measure the absorbance at 540
Incubate the plate for 30 min in the dark at 28 °C.
nm in a plate reader.
4. Measure the fluorescence in a fluorescence plate
NOTE: A second readout at 690 nm should be
reader at 530 nm (excitation) and 595 nm (emission)
carried out to exclude any background fingerprint
for AB, and at 493 nm (excitation) and 541 nm
absorbance in the plate.
(emission) for CFDA-AM.
3. MTT assay
2. NR assay
NOTE: The MTT assay must be carried out separately
NOTE: The steps for the NR assay are carried out
from the assays described above (in a new plate) (Figure
immediately after the AB and CFDA-AM assays (Figure
2).
1).
1. Carefully remove the exposure media by pouring the
1. Centrifuge the NR working solution (40 µg/mL) at
content into a collection tray.
600 × g for 10 min.
2. Add 100 µL of MTT working solution per well using a
NOTE: Precipitation of NR in the tube must
multichannel micropipette. Incubate the plate at 28
not be transferred to the plates. Thus, after
°C for 4 h.
centrifugation of the NR working solution, collect
the supernatant using a pipette without aspirating 3. Discard the MTT solution by pouring the content into
the NR precipitates. Transfer the supernatant to a a collection tray.
reagent reservoir.
NOTE: Absorbance (abs) or fluorescence (fluo) units
4. Add 100 µL per well of DMSO to extract the
represent the mean of absorbance or fluorescence
formazan crystals, incubating the plate on a plate
measured in the three wells per concentration; blank
shaker for 10 min. Measure the absorbance at 570
represents wells without cells.
nm using a plate reader.
NOTE: A second readout at 690 nm should be
Representative Results
carried out to exclude any background fingerprint
absorbance in the plate. It is important to note Figure 3 shows the plates of the AB, CFDA-AM, NR, and
that test chemicals may interfere with MTT, which MTT assays. For the AB assay (Figure 3A), the blank
must be evaluated to ensure the quality of the wells and wells with no or a reduced number of viable
cells show blue color and low fluorescence, while the
generated data32 . For this, cell-free wells containing
wells with a high number of viable cells are pinkish and
the test concentrations and MTT (0.5 mg/mL) should
present high fluorescence values due to the transformation
be exposed, followed by incubation to observe
of resazurin (AB) into resorufin (pinkish substance) by the
any color change in the wells that may increase
viable cells. For the CFDA-AM assay, there is no visible
the absorbance and lead to false viability results.
difference in the color of the wells on the plate; however, the
Chemicals that interact with MTT must be avoided
fluorescence is higher in wells containing viable cells due to
in this test.
the retention of CFDA-AM and subsequent conversion into
6. Calculating cell viability/cytotoxicity carboxyfluorescein (fluorescent substance).
NOTE: The raw absorbance or fluorescence acquired is For the NR assay (Figure 3B), the blank wells must be
used to calculate cell viability as a percentage related to transparent with very low absorbance values since there
the negative control (for test chemicals prepared directly are no cells to retain the NR dye. In some cases, the
in exposure media) or solvent control (for test chemicals blank wells are not transparent, indicating the occurrence of
prepared using solvents, such as DMSO). Before determining NR precipitation on the plate; in this instance, this should
the cell viability percentage, the raw data must be normalized not be considered a valid experiment. Highly cytotoxic
by the blank control. concentrations of the test chemicals and PC are transparent
or present a very light pink color with low absorbance values,
1. Calculate the average absorbance or fluorescence for
while wells containing viable cells retain the NR dye and
each test chemical concentration and control group
present a dark pink color and high absorbance values.
(three wells/treatment).
For the MTT assay (Figure 3C), the blank wells must be
2. To determine the cell viability percentage relative to
transparent and with very low absorbance as there are
control (negative or solvent), use equation (2):
no cells to convert MTT into formazan. Highly cytotoxic
concentrations of the test chemicals and PC are transparent
(2) or present a very light violet color with low absorbance values,
while wells containing viable cells transform the MTT (yellow) with at least three technical replicates and three experimental
into formazan (violet substance), presenting a darker violet replicates using nominal concentrations In the AB assay. The
color with high absorbance values. analyses were performed with five different concentrations
of the test substances; however, a higher number of
Figure 4A shows a representative graphic of cell viability
concentrations may be required depending on the type
after the calculation, using the averages of fluorescence or
of experiment. For instance, eight test concentrations are
absorbance values per group. The graphic can be plotted
recommended for the range-finding test, which is usually
with the input of viability percentage values, calculated by the
performed to determine final test concentrations of a chemical
viability calculation formula presented in protocol section 6,
substance for an experiment. As the viability assays have
using data analysis software. The viability of cells exposed
different cytotoxicity endpoints, we recommend calculating
to the SC should not be 10% lower than in the NC17 . The
the IC50 for each assay performed separately to identify
cell viability percentage for the test chemicals is calculated
differences in sensitivity caused by different mechanisms of
related to the NC or SC, depending on their solubility. In this
action of the chemical substances or differences in sensitivity
case, different concentrations of DMSO are used as a test
among cell lines. The differences in IC50 values of tested
substance and the cell viability is related to NC, which is
chemicals in different cell lines may also vary depending
defined as 100% viability.
on the type of culture medium used, since differences in
The cell viability data can be used to calculate the test composition related to proteins and lipids can impact the
chemicals' half maximal inhibitory concentration (IC50) by chemical bioavailability36 . In addition, the cytotoxicity of many
logarithmic transformation, and interpolated standard curve chemicals can be evaluated through these assays. The IC50
by nonlinear regression after appropriate replicates33 , 34 , 35 . of other chemicals evaluated in ZFL and ZEM2S cell lines is
Figure 4B shows the IC50, calculated from the viability shown in Figure 4B-H.
percentage shown in Figure 4A. The IC50 was obtained
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 the chemical exposure condition, and maximal acceptable
click here to download this Table. concentration for the SC). As these assays quantify
cytotoxicity by different cell viability endpoints (metabolic
Discussion
function, lysosomal membrane integrity, and cell membrane
Cytotoxicity assays are widely used for in vitro toxicity
integrity), the combination thereof provides an accurate
evaluation, and this protocol article presents four commonly
evaluation of chemical cytotoxicity in zebrafish cell lines. This
used cytotoxicity assays modified to be performed in
protocol also recommends the culture of ZFL and ZEM2S
zebrafish cell lines (i.e., cell density for 96-well plate,
cell lines in a CO2-free condition, due to their culture media
incubation time in the MTT assay, FBS depletion during
composition that can adequately buffer the culture system,
maintaining pH at 7.4 (physiological pH). The culture media deprivation might depend on the type of the test chemical.
composition and the CO2-free environment proposed in this For example, Pomponio et al.46 reported that although the
protocol for both cell lines are widely reported in the literature. chemical amiodarone has a higher binding to plastic in
The ZFL cell line is usually cultured in L-15 and RPMI media the absence of FBS, its bioavailability is even lower when
with or without the addition of sodium bicarbonate and without using 10% FBS. For mono-N-desethylamiodarone, almost the
CO237 , 38 , 39 , 40 , 41 , 42 , 43 . Meanwhile, the ZEM2S cell line is same amount is bound to the serum medium and to the
cultured according to instructions of the bioresource center, walls46 .
and its culture media is formulated for CO2-free cultures; thus,
Organic solvents (e.g., DMSO) are generally recommended
CO2 and air mixture can be detrimental to cells when using
to be used up to 0.5% in in vitro assays. However, this
this type of culture media44 .
low concentration can impair testing higher concentrations
The chemical exposure in a culture medium without adding of poor water-soluble chemicals. To prevent this issue, we
FBS was performed based on published studies, showing evaluated whether higher concentrations of DMSO were
that the chemical substances' bioavailability in in vitro assays suitable for ZFL and ZEM2S cell lines not exceeding the
is significantly impacted by their binding to serum proteins. cytotoxicity threshold of 10%. For this, non-treated cells
For instance, Chen et al.45 showed that the presence of (NCs) and cells treated (24 h) with different concentrations
serum proteins in the RTgill-W1 assay could reduce the of DMSO (0.1%, 0.5%, and 1%) were processed for the
bioavailability of a cationic surfactant (C12-benzalkonium) MTT and NR assays. The results showed no significant
up to three-and-a-half-fold. The chemical bound to FBS difference in cell viability from the treatment group compared
generally ranged from 47% to 90% in the culture medium45 . to NCs (Figure 6), indicating that, under these particular
Thus, to avoid this issue, we evaluated the cell viability of conditions, concentrations of DMSO up to 1% can be used.
ZFL and ZEM2S cells in cultures completely deprived of Other fish cell lines also support DMSO concentrations higher
FBS for 24 h using the MTT assay. The results showed than 0.5%, which is not a particularity of ZFL and ZEM2S
no significant difference in cell viability from ZFL or ZEM2S cell lines. For instance, maximal solvent concentrations of
cultures with (10%) and without (0%) FBS, indicating that up to 2% DMSO (with no cytotoxic effect observed) have
these zebrafish cell lines can be subject to chemical treatment been applied in cytotoxicity assays using CHSE-214 (cell
in culture media deprived of FBS (Figure 5). It is important line derived from Oncorhynchus tshawytscha embryo)47 ,
to highlight that decreasing the amount of FBS might cause RTG-2 (Oncorhynchus mykiss gonadal cell line)48 , 49 , 50 , and
other consequences in chemical bioavailability. For instance, PLHC-1 (Poeciliopsis lucida hepatocellular carcinoma cell
lipophilic chemicals have higher sorption to plastic labware line)48 cells. The maximum solvent concentration of 1%
and plates, reducing chemical bioavailability36 . However, the DMSO has also been used in cytotoxicity assays using RTL-
presence of FBS in culture medium can decrease plastic W1 (Oncorhynchus mykiss gonadal cell line)51 and CCO
binding due to serum constituents competing with the plastic (Ictalurus punctatus ovary cell line)52 cells. According to
for binding to the chemicals46 . The best decision related Mori and Wakabayashi47 , fish cell lines may have a lower
to the percentage of FBS supplementation or its complete sensitivity to DMSO than mammalian cell lines. However,
it is important to highlight that the maximal acceptable This protocol can be used to study the effects of chemical
concentration of 1% DMSO was exclusively defined for substances on fish by using in vitro models. Different cell lines
cytotoxicity, and this should be carefully evaluated for other may have different sensitivities to estimate chemical effects,
endpoints (e.g., genotoxicity, epigenetics, protein-coding even though they are from the same group of vertebrates,
gene expression analysis) in ZFL and ZEM2S cell lines. such as fish. For instance, comparing ZFL and ZF4 cell lines
with fish embryos, Langu-Mitea et al.21 demonstrated that
The MTT and AB assays are based on metabolic activity
ZFL is capable of generating similar results compared to the
to determine cell viability. Although the MTT assay is
fish embryo toxicity test. Tanneberger et al.5 showed that
the most used viability assay, in comparison to the AB
the permanent fish cell lines GSF, PLHC, RTG-2, RTgill-
assay it can be slightly less sensitive, overestimating cell
W1, and R1 have different sensitivities in predicting chemical
viability in some cases53 . The higher sensitivity of the
fish toxicity compared to in vivo (adult fish). Although RTgill-
AB assay may be related to the measurement method,
W1 (permanent fish gill cell line) was recently validated
as fluorescence measurement is more sensitive than
as an alternative method to predict fish acute toxicity, the
colorimetric measurement15 . Nonetheless, both the AB and
applicability of other cell lines in ecotoxicity studies should
MTT assays are high-quality assays to identify cytotoxic
be investigated. Using cell lines from different fish species
chemical substances, and their generated data have been
and tissue origins as well as developmental stages (ZEM2S:
used to classify hazardous chemical substances according to
embryo; ZFL: adult liver) may significantly contribute to
their intrinsic cytotoxic potential53 .
ecotoxicity studies, since it can address effects related to
The idea of performing the AB, CFDA-AM, and NR assay specific stages of fish development and target organs. Thus,
in the same plate was based on the OECD TG 249 (RTgill- chemical effects should be investigated using different cell
W1 assay)17 ; however, modifications were made to perform cultures reflecting different target sites in fish (e.g., liver,
these assays in 96-well plates, as well as to be suitable gonads, gill, brain), and not only in a single cell line55 .
for zebrafish cell lines. Assays in 96-well plates, instead

These protocols can have different applications in ecotoxicity
of 24-well plates, can be advantageous for high-throughput
studies, and their use is not necessarily limited to predicting
cytotoxicity testing in fish cell lines. In addition, the RTgill-
fish acute toxicity. For instance, they can be applied to
W1 assay uses only L-15 culture medium, while the ZFL and
define sub-cytotoxic concentrations to perform other in vitro
ZEM2S cell lines are cultured in a medium containing D-
fish toxicity testing, such as evaluating endocrine-disrupting
glucose. The culture medium makes it possible to perform the
effects on fish. In addition, the in vitro cytotoxicity data
MTT assay in these cell lines, as cell lines cultured in glucose-
can also help develop and improve physiologically-based
free medium (e.g., only L-15) may immediately decrease the
toxicokinetic (PBTK) modeling. As PBTK focuses on the
MTT reduction in cells and impair the performance of this
distribution of a chemical in an organism considering the
assay54 . This protocol allows four different viability assays to
different organs (such as the gill, liver, or intestine)56 , using
be easily performed in two zebrafish cell lines.
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 4. Scholz, S. et al. Alternatives to in vivo tests to
data representing biological processes in the PBTK model detect endocrine disrupting chemicals (EDCs) in fish
and contribute to in vitro to in vivo extrapolation21 , 56 . and amphibians-screening for estrogen, androgen
and thyroid hormone disruption. Critical Reviews in
Disclosures
Toxicology. 43 (1), 45-72 (2013).
The authors declare no conflict of interest.
5. Tanneberger, K. et al. Predicting fish acute toxicity using
a fish gill cell line-based toxicity assay. Environmental
Acknowledgments
Science & Technology. 47 (2), 1110-1119 (2013).
In memory of Dr. Márcio Lorencini, a coauthor of this work,
6. Roesler, R., Lorencini, M., Pastore, G. Brazilian cerrado
an excellent researcher in the field of cosmetics and devoted
antioxidant sources: cytotoxicity and phototoxicity in
to promoting cosmetic research in Brazil. The authors are
vitro. Food Science and Technology. 30, 814-821 (2010).
grateful for the Multi-user Laboratory in the Physiology
Department (UFPR) for equipment availability and for the 7. Ruyra, A. et al. Zebrafish liver (ZFL) cells are able to
financial support of the Coordination for the Improvement of mount an anti-viral response after stimulation with Poly
Higher Education Personnel (CAPES, Brazil) (Finance Code (I:C). Comparative Biochemistry and Physiology. Part B,
001) and the Grupo Boticario. Biochemistry & Molecular Biology. 182, 55-63 (2015).
8. Natsch, A., Laue, H., Haupt, T., von Niederhäusen,

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Cytotoxic and genotoxic effects of water and sediment
samples from gypsum mining area in channel catfish

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Cytotoxicity Assays with Zebrafish Cell Lines

characteristics of liver parenchymal cells and can metabolize

Protocol number of viable cells using equation (1):

4. Transfer the cell suspension to a 15 mL conical centrifuge

1. AB and CFDA-AM assays microscope. NR precipitates may interfere with the

the NR precipitates. Transfer the supernatant to a a collection tray.

percentage shown in Figure 4A. The IC50 was obtained

for zebrafish cell lines. Assays in 96-well plates, instead

8. Natsch, A., Laue, H., Haupt, T., von Niederhäusen,

and Biocidal Products Regulations. Available at 931-941 (2018).

<87ebb68f-2038-f597-fc33-f4003e9e7d7d> (europa.eu). 9. Freshney, R. I. Cytotoxicity. Culture of Animal Cells: A

50. Hollert, H., Duerr, M., Erdinger, L,. Braunbeck, T.

51. Pannetier, P. et al. Toxicity assessment of pollutants

52. Ternjej, I., Srček, V. G., Mihaljević, Z., Kopjar, N.

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