An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines.

Abstract

The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the existing legislation and the approval of the new European law regarding the production and importation of chemicals, known as REACH. This new legal framework supports the development of alternative methods to animal experimentation encouraging the improvement and/or design of new methodological strategies for the toxicological evaluation of chemical compounds. Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best agreement between obtaining relevant information about the mechanism of toxic action and the use of alternative methods. Following this trend, this work presents an integrated cellular strategy in order to know the toxicity and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The experimental procedures are performed in two steps. The first one involves the systematic analysis of the main cellular targets using proliferation, viability and morphological probes. The second step relies upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic action and the cellular response. The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.

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An integrated cellular model to evaluate cytotoxic effects in mammalian cell lines
P. Fernández Freire, A. Peropadre, J.M. Pérez Martín, O. Herrero, M.J. Hazen*
Cellular Toxicology Group, Department of Biology, Universidad Autónoma de Madrid, Spain
a r t i c l ei n f o
Article history:
Received 29 October 2008
Accepted 15 June 2009
Available online 18 June 2009
Keywords:
Cellular model
In vitro
Cytotoxicity
a b s t r a c t
The ever growing anthropogenic pressure to the environment has lead in 2007 to the revision of the exist-
ing legislation and the approval of the new European law regarding the production and importation of
chemicals, known as REACH. This new legal framework supports the development of alternative methods
to animal experimentation encouraging the improvement and/or design of new methodological strate-
gies for the toxicological evaluation of chemical compounds.
Even though cytotoxicity studies are a reductionist approach to acute toxicity in vivo, they offer the best
agreement between obtaining relevant information about the mechanism of toxic action and the use of
alternative methods.
Following this trend, this work presents an integrated cellular strategy in order to know the toxicity
and mechanism of action of chemical compounds, using simple and reproducible in vitro systems. The
experimental procedures are performed in two steps. The first one involves the systematic analysis of
the main cellular targets using proliferation, viability and morphological probes. The second step relies
upon the results obtained in the first step, including specific assays that focus on the mechanism of toxic
action and the cellular response.
The benefits of this strategy are exemplified with two real cases: pentachlorophenol and rotenone.
? 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The Earth is nowadays threatened by an ever growing anthro-
pogenic pressure, mainly due to the leakage of chemical
compounds to the environment. Extensive exposure to these com-
pounds leaded to the revision of the existing legislation and a new
European law, known as REACH, came into force in 2007 (EC,
2006). Due to the great quantity of toxicological studies that this
situation requires, the new legal framework supports the develop-
ment of alternative methods to animal experimentation according
to the philosophy of the 3 Rs. This circumstance represents an
exceptional chance for the improvement and/or design of new
methodological strategies for the toxicological evaluation of chem-
ical compounds (Hengsteler et al., 2006).
In this context, cell cultures turn out as a particularly interest-
ing field, considering the wide spectrum of existing methodologies
suitable for toxicological evaluation. Assuming that toxic effects
seen in a whole organism are due to a prior failure of basic cellular
functions (Eisenbrand et al., 2002; Walum, 1998), cytotoxicity
studies offer a good compromise between using alternative meth-
ods and elucidating the mechanism of toxic action, even though
they are a reductionist approach to acute toxicity in vivo.
According to the guidelines of international organisms (ICC-
VAM, 2003), this study proposes a tiered cellular approach in
which proliferation, viability and morphological endpoints are
combined in order to acquire fast and reproducible results,
bringing together Toxicology and Cell biology and obtaining as
much information as possible about the behavior of chemical
compounds in mammalian cells. Two well-known environmental
pollutants whose mechanism of toxic action is not fully understood
(pentachlorophenol and rotenone), and three different continuous
cell lines routinely used in toxicological studies (Vero, HeLa, 3T3)
were selected in order to demonstrate the suitability of the exper-
imental approach.
2. Materials and methods
2.1. Chemicals
Pentachlorophenol (PCP, CAS 87-86-5), and rotenone (ROT, CAS
83-79-4) were purchased from Sigma Chemical Co. (St. Louis, USA).
Stock solutions were diluted in ethanol 100% (PCP) or dimethyl
sulfoxide (ROT) and stored in darkness at room temperature.
2.2. Cell culture and treatments
Three different mammalian cell lines were selected to perform
the experiments: HeLa (human adenocarcinoma), 3T3 (mouse
0887-2333/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2009.06.017
* Corresponding author. Address: Departamento de Biología, Facultad de Cien-
cias, Universidad Autónoma de Madrid, C/Darwin 2, 28049 Madrid, Spain. Tel.: +34
914978248.
E-mail address: mariajose.hazen@uam.es (M.J. Hazen).
Toxicology in Vitro 23 (2009) 1553–1558
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embryonic fibroblasts) and Vero (monkey kidney fibroblasts). Cell
cultures were routinely maintained at 37 ?C in a 5% CO2humidified
atmosphere using Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with fetal calf serum (5% for Vero and 3T3, 10% for
HeLa), penicillin (100 U/ml), streptomycin (100 mg/ml) and L-glu-
tamine (2 mM). All reagents for cell cultures were products of Lon-
za (Basel, Switzerland).
Exponentially growing cells were seeded at a density of 105
cells/ml into 24-well tissue culture plates (BD FalconTM, California,
USA) and cultured in complete medium for 24 h before adding
the treatments. After washing the monolayers once in PBS, work-
ing solutions for each compound (PCP 1 – 100 lM; ROT 0.2 –
1 lM) were added by indirect dosing after sterilization by filtration
through a 0.22 lm Millipore?filter. Solvent concentration in med-
ium was lower than 0.5% (for DMSO) or 1% (for ethanol) including
the control groups.
2.3. Experimental design
The experimental procedures for the toxicological evaluation
were scheduled in a step-wise approach in order to gain the higher
amount of information for each compound (Fig. 1).
The first step entails the systematic analysis of the main cellular
targets for basal cytotoxicity by means of quantitative and qualita-
tive assays. The battery of classical quantitative tests performed in-
cluded Total Protein Content (TPC) to evaluate cell proliferation
(Bradford, 1976), MTT reduction to measure mitochondrial dys-
function (Mosmann, 1983) and Neutral Red Uptake (NRU) to assess
membrane stability (Borenfreund and Puerner, 1985).
Complementary morphological studies allowed us to evaluate
the integrity of key cellular structures and organelles using differ-
ent microscopic techniques as previously described (Labrador
et al., 2007): toluidine blue staining for general morphology;
in vivo fluorescent probes for mitochondrial reticulum (rhodamine
123) and endosomal compartment (acridine orange); specific
detection of actin microfilaments (phalloidin – TRITC); and indirect
immunofluorescence for the microtubule network (anti a tubulin –
FITC). All the reagents but acridine orange (BDH, Dorset, UK) were
purchased from Sigma.
The second step involves different experimental procedures
depending on the results obtained in the first step, focusing on
the determination of the mechanism of toxic action and the
cellular response. The precise methods will vary depending on
the results obtained for each compound in the first step (plasma
membrane integrity, cell cycle progression, mitochondrial physiol-
ogy, cytoskeleton integrity, endomembrane system, etc.). For the
compounds tested in this study the evaluation of nuclear morphol-
ogy with Hoechst 33258 (Fernández Freire et al., 2005), mitotic in-
dex scoring (Pérez Martín et al., 2008), and relocalization of
acridine orange (Yuan et al., 1997); as well as specific immunoflu-
orescence for cell death-related proteins like cytochrome c (Cyt C)
and apoptotic inducing factor (AIF), performed following the
instructions of the manufacturer (Santa Cruz Technologies, USA)
were included.
2.4. Data analysis
Experiments were performed at least three times and each con-
centration group was assayed using triplicated wells. Concentra-
tion–response cytotoxicity curves were generated with individual
data points expressed as percentage of that found in untreated cul-
tures, and presented as the arithmetic mean ± standard deviation,
using Microsoft?Excel 2007. Statistical analysis, including analysis
of variance (ANOVA) with the appropriate post hoc test (Bonferroni
or Games-Howell) and non-linear regression for the determination
of the IC10and IC50values, were carried out using GraphPad Prism
4.0 for windows (GraphPad Software Inc., USA). The level of statis-
tical significance was in all cases p 6 0.05.
3. Results
3.1. First step: basal cytotoxicity
3.1.1. Quantitative assays
The effect of 24 h exposure to growing concentrations of PCP
and ROT on cell proliferation (TPC) and viability (MTT, NRU) of
the three cell lines is shown in Fig. 2 and the IC10and IC50values
are presented in Table 1. The toxicological pattern remains quite
similar after PCP treatments independently of the cell line tested,
but there are some interesting differences. Vero cells are the first
ones detecting significative differences with the control in all the
parameters evaluated, even though MTT assay with HeLa cells
showed higher toxicity level. Despite this circumstance, concentra-
tion–response curves and IC10values reveal that Vero cells were
much more sensitive than HeLa and 3T3 after PCP treatments.
As far as ROT exposure is concerned, the sensitivity of Vero cell
line is highlighted with all the endpoints tested, while HeLa cell
line showed a mild toxicity pattern and 3T3 cell line was not se-
verely affected.
The comparison between different endpoints in Vero cell line
after PCP exposure detected significant differences from the lowest
concentration tested (1 lM) only with MTT reduction test. Not-
withstanding this circumstance, the IC50value for NRU assay was
considerably lower than those obtained with the other two
endpoints.
On the other hand, concentration–response curves for ROT dis-
played a similar trend independently of the parameter evaluated,
while the lowest IC values were achieved with the TPC assay.
On the whole, the results obtained with this quantitative assays,
suggest a specific damage in the cellular membranes after PCP
treatments, and alterations affecting the proliferation or adhesion
of cells exposed to ROT.
3.1.2. Morphological studies
For both compounds, the first concentration with statistically
significant differences (5 lM for PCP and 0.2 lM for ROT) was se-
lected to perform the microscopic observations only on Vero cells.
Representative images of cellular organelles and structures
after exposure to PCP and ROT can be seen in Fig. 3. General mor-
phology analyzed after toluidine blue staining reveal minor
Fig. 1. Scheme of the experimental design indicating the arrangement of the
different experimental procedures included.
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changes after 24 h treatment with PCP 5 lM, but severe cellular
retraction and rounding up could be observed after exposure to
ROT 0.2 lM. Compared with the filamentous mitochondrial net-
work in control cells, treatment with both compounds lead to frag-
mentation, even though it was much more severe with PCP than
with ROT. Acridine orange uptake demonstrated a mild swelling
of lysosomes along with an increased green fluorescence of the
cytoplasm after PCP exposure. As far as ROT exposure is concerned,
no significant disturbances, apart from those derived of the cellular
shrinkage, were observed after 24 h.
The integrity of the cytoskeleton was not affected by the treat-
ments with low concentrations of PCP, with stress fibers clearly
visible and no changes in the microtubule network. On the other
hand, when Vero cells were subjected to ROT exposure, the integ-
rity of the microtubules was highly compromised, even though
there was no clear effect upon the actin microfilaments.
3.2. Second step: mechanism of action and cellular response
3.2.1. Pentachlorophenol
The most remarkable effect detected after the exposure of Vero
cells to PCP during 24 h in the first experimental step was the dis-
ruption of the endosomal compartment, showed by quantitative
(NRU) and qualitative (AO staining) endpoints. Thereby, in this sec-
ond step we performed some specific experiments to determine
more precisely the mechanism of action involved in the toxicity
of PCP.
The AO relocalization assay is able to identify early injuries in
the endosomal compartment by means of variations upon the
green fluorescence of the cell. The performance of this test with a
low concentration of PCP (5 lM) at three different time points (3,
4 and 6 h) is shown in Fig. 4. Even from the earliest exposure time
evaluated, a significant increase in the green fluorescence intensity
Fig. 2. Concentration–response curves after 24 h exposure of Vero (continuous line, closed circle), HeLa (dotted line, open square) and 3T3 (discontinuous line, closed
triangle) cells to PCP and ROT. Statistically significance with the control (p 6 0.05) for each cell line is represented by its own symbol.
Table 1
Comparison of EC values obtained for the three mammalian cell lines studied after PCP and ROT treatments.
Cell lineEndpointPCP (lM)ROT (lM)
IC10IC50IC10IC50
Vero TPC
MTT
NRU
0.86
0.42
2.28
27
11.89
6.77
0.01
0.04
0.09
0.39
0.43
0.66
HeLaTPC
MTT
NRU
4.61
5.02
4.39
8.25
6.88
6.4
0.42
0.56
0.23
0.73
0.84
0.45
3T3TPC
MTT
NRU
3.76
9.94
5.42
11.73
11.86
7.76
0.2
0.41
0.48
>1
>1
>1
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of the cytoplasm was detected. This fact, together with the absence
of mitochondrial fragmentation under these same experimental
conditions, indicates an early and specific disturbance of the endo-
somal compartment.
In this same line, and in order to determine if the lysosomal
damage was in fact affecting mitochondrial integrity, specific
inmunostaining against Cyt C and AIF were performed. The distri-
bution of AIF in control cells was congruent with mitochondrial
localization. However, after exposure to PCP 5 lM during 24 h,
the pattern displayed was consistent with translocation into the
nucleus. Under the same experimental conditions, Cyt C remained
in the mitochondria as in untreated cells (Fig. 5). This circumstance
suggests a specific permeabilization of the outer mitochondrial
membrane.
3.2.2. Rotenone
As the results of the first part indicates a possible interference of
ROT with the proliferation and/or adhesion properties of the cul-
tured cells, a fast and easy test as the mitotic index scoring was
performed after 4, 8 and 24 h. A sudden significant increase in
the division rate, due to the amount of metaphases, was detected
with the shorter exposure time, reaching its maximum after 8 h
(Fig. 6A). From 8 to 24 h, the number of metaphases decreased,
while in parallel an equivalent number of multinucleated and dead
cells was detected (Fig. 6A and B).
4. Discussion
The integrated cellular model proposed in this study has proved
to be highly effective to evaluate the toxicity of chemical com-
pounds. In the first place, the assessment of basal cytotoxicity end-
points with Vero, HeLa and 3T3 cells demonstrated the suitability
of the selected battery of quantitative tests to be performed with
different mammalian cell lines. In addition, this approach allowed
us to compare their sensitivity to detect toxic effects. The lowest
IC10values obtained with Vero cells and their suitability for mor-
phological studies signaled them as our choice to carry out all
the remaining experimental procedures.
On the other hand, the advantages of a well-planed experimen-
tal design are highlighted by the interesting information acquired
on each stage. The combination of proliferation and specific viabil-
ity assays is a quick way to decide the primary cellular targets of a
chemical compound, while complementary morphological analysis
are useful either to confirm (endosomal compartment for PCP), to
point out interesting side-effects (Labrador et al., 2007), and/or to
show new unexpected effects (microtubule network for ROT).
Applying this experimental approach, the quantitative cytotoxic
assays pointed to a preferential effect on the endosomal compart-
ment for PCP, and to an interference of ROT either with the prolif-
eration machinery or the adherent properties of the cells.
The disturbance of the endosomal compartment of Vero cells ex-
posed to PCP has previously been suggested (Fernández Freire et al.,
2005), but here we have been able to determine the precise under-
lying mechanism of toxic action. The results obtained with the
highly specific acridine orange relocalization test confirmed the
early disruption of the endosomal/lysosomal membranes. The par-
tial leakage of the content of these organelles has been lately
relatedwiththeinitiationoftheso-calledlysosomalapoptoticpath-
way (Bidere et al., 2003), which requires the early permeabilization
of the lysosomal membranes and the later disruption of the mito-
chondria (Kroemer et al., 2007). In this circumstances, the mito-
chondrial injury is mainly due to the selective permeabilization of
the outer mitochondrial membrane (Galluzzi et al., 2007), which
can be demonstrated, as in our case, by the release to the cytoplasm
of certain apoptotic factors located in the intermembrane space
Fig. 3. Systematic analysis of the main morphological targets of Vero cells after a 24 h treatment with PCP 5 lM or ROT 0.2 lM. From left to right: general morphology,
mitochondria, endosomal compartment, actin microfilaments and microtubule network. Bar = 10 lm.
Fig. 4. Changes in the green fluorescence intensity after different time–length
exposures to PCP 5 lM obtained with the AO relocalization technique.
the beginning of statistically significant differences with the control.
Indicates
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(AIF), while other more tightly anchored (Cyt C), remain inside the
mitochondria.
Concerning ROT toxicity, the general toxic effect detected on the
first experimental step was further investigated with proliferation-
related tests. The mitotic index scoring was enough to demonstrate
a strong effect upon the normal cell cycle progression along time
due to a severe cell cycle arrest in prometaphase, as has been pre-
viously described (Armstrong et al., 2001; Srivastava and Panda,
2007). This effect was switched onto nuclear morphology changes
when the exposure time reached 24 h. The increase in multinucle-
ated and dead cells correlated with the decrease of metaphases in
this time point, which is the hallmark to identify the undergoing
toxic effect as a mitotic catastrophe (Castedo et al., 2004).
On the whole, the interesting results presented in this study are
a probe of the usefulness of the proposed experimental approach.
The use of Vero cells for toxicological assays and the scheduled
of a common set of tests, including morphological studies, followed
by specific assays should be encouraged and taken into consider-
ation for possible future validation processes.
Acknowledgements
This work was supported by the Spanish Ministry of Science
and Innovation (CTM2005-02135) and the Community of Madrid
(CG06-UAM/AMB-0186). A. Peropadre is a recipient of a contract
from the Community of Madrid (Contrato de Personal Investigador
de Apoyo), and J.M. Pérez Martín of a grant from the Universidad
Autónoma de Madrid (Formación Personal Universitario).
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