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Induction of apoptosis in mussel Mytilus galloprovincialis gills by model

cytotoxic agents

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DOI: 10.1007/s10646-011-0746-6 · Source: PubMed

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Induction of apoptosis in mussel Mytilus
galloprovincialis gills by model cytotoxic
agents

A. Châtel, B. Hamer, Ž. Jakšić,

V. Vucelić, H. Talarmin, G. Dorange,
H. C. Schröder & W. E. G. Müller

Ecotoxicology

ISSN 0963-9292
Volume 20
Number 8

Ecotoxicology (2011) 20:2030-2041

DOI 10.1007/s10646-011-0746-6

1 23
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 Author's personal copy
Ecotoxicology (2011) 20:2030–2041
DOI 10.1007/s10646-011-0746-6

Induction of apoptosis in mussel Mytilus galloprovincialis gills

by model cytotoxic agents
A. Châtel • B. Hamer • Ž. Jakšić • V. Vucelić •

H. Talarmin • G. Dorange • H. C. Schröder •

W. E. G. Müller

Accepted: 12 July 2011 / Published online: 30 July 2011

Ó Springer Science+Business Media, LLC 2011

Abstract Apoptosis signaling pathway was investigated
in the marine mussel Mytilus galloprovincialis exposed to
various stressors. Analyses were performed in mussels
exposed to two major pollutants of the aquatic environ-
ment: tributyltin and the water soluble fraction of diesel oil,
for 1 h and animals were then maintained in sea water for a
recovery period of 6 and 24 h. Apoptosis was evaluated at
several levels of the cell signaling cascade by measuring
Bcl-xS expression, caspase-3 activity and DNA damage
(Fast micromethodÒ and TUNEL techniques). H2O2 was
used as a control of apoptosis induction for validation of
the assays. Results showed an induction of Bcl-xS
expression, a protein implicated in apoptosis, after 1 h
exposure to all concentrations of chemicals. Moreover, in
the same manner, apoptotic DNA damage was induced
with all chemicals tested. Besides, caspase 3 activity was
detected after 1 h exposure to low doses of TBT and diesel
oil while the high concentrations induced this protein after
6 h. The achieved data were also correlated with our pre-
vious study, demonstrating an induction of the mitogen-
activated protein kinase (MAPK) activity in the mussel
M. galloprovincialis exposed to the same conditions. In
conclusion, this study was one of the first characterizing the
MAP kinase cell signaling pathway leading to apoptosis in
the mussel M. galloprovincialis exposed to chemicals. It
showed for the first time that the Bcl-xS protein was
present in these mussels as in other species and played a
role in apoptosis mediation. Moreover, the main originality
of this work was that it showed that two apoptotic path-
ways might be present in the mussel: a caspase 3-depen-
dent and a caspase 3-independent pathways.
Keywords Biomarker
Apoptosis
Bcl-xS
Caspase

DNA damage
Mytilus galloprovincialis
Introduction

A. Châtel (&)
H. Talarmin
G. Dorange

EA 4326 Facteurs nerveux et structuration tissulaire, Université
de Bretagne Occidentale, 22 avenue Camille Desmoulins, 29609
Brest cedex, France
e-mail: amelie.chatel@cemagref.fr
A. Châtel
H. C. Schröder
W. E. G. Müller
Institute for Physiological Chemistry, Johannes Gutenberg
University, Duesbergweg 6, 55099 Mainz, Germany
B. Hamer
Ž. Jakšić
Rud̄er Bošković Institute, Center for Marine Research,
Laboratory for Marine Molecular Biology, Giordano Paliaga 5,
HR-52210 Rovinj, Croatia
V. Vucelić
Teaching Institute of Public Health, Primorsko-Goranska
County, Medical Faculty, University of Rijeka, HR-51000
Rijeka, Croatia
Apoptosis, or programmed cell death, is a fundamental and
complex biological process that enables an organism to get
rid of damaged cells during development, normal homeo-
stasis and disease (Porter and Janicke 1999). It can also be
initiated by extracellular agents such as hormones, cyto-
kines, as well as chemical, physical and viral agents.
The apoptotic mechanism is characterized by morpho-
logical changes including chromatin condensation, nuclear
fragmentation and appearance of apoptotic bodies (Kerr
et al. 1972; Wyllie et al. 1980), as well as biochemical
changes such as degradation of DNA first into large frag-
ments (50–300 kbp) and subsequently into smaller frag-
ments so-called oligonucleosomes (Wyllie et al. 1980;
Schwartzman and Cidlowski 1993; Zheng et al. 1998;
Enari et al. 1998).

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Induction of apoptosis in mussel Mytilus galloprovincialis
Signaling of apoptosis occurs through multiple inde-
pendent pathways that are initiated either from triggering
events within or from outside the cell: the extrinsic and
intrinsic pathways. The first one involved activation of
death receptor whereas the second way implies the mito-
chondria. All apoptosis signaling pathways converge on a
common machinery of cell destruction that is activated by
a family of cysteine proteases (caspases), the effector
proteins of apoptosis which cleave substrate at aspartate
residues. The caspases stand out as being crucial in diverse
metazoan organisms (Nicholson and Thornberry 1997).
Although the critical role of caspases in apoptosis was
first demonstrated by the isolation of the ced-3 gene in the
nematode Caenorhabditis elegans (Yuan et al. 1993),
caspases have been cloned in mammals and Drosophila
(Fraser and Evan 1997; Salvesen and Dixit 1997). Caspases
are highly conserved proteins since they are known to be
activated in the oyster Crassostrea gigas in response to
b-adrenergic signaling (Lacoste et al. 2001). Recently,
Kefaloyanni et al. (2005) showed a strong activation of
caspase 3 activity in the mantle of Mytilus galloprovin-
cialis after exposure to Cu2?, Zn2? and Cd2? and dem-
onstrated that SB203580, an inhibitor of p38 MAPK,
abolished the activation of caspase 3 induced by Cu2?, but
a some informations were missing concerning their activity
in molluscs.
Apoptosis is also regulated by several elements and in
particular, by the family of polypeptides homologous to
Bcl2 which plays an important role in this process. Among
the proteins of this family, Bcl-x is located predominantly
in mitochondrial membranes.
The Bcl-x gene has three alternative splice forms,
bcl-xL(a), bcl-x(b) and bcl-xS(c) (Boise et al. 1993;
Gonzalez-Garcia et al. 1994). Previous studies have shown
that Bcl-xS was a pro-apoptotic protein which, in stable
transfectants, inhibits the ability of Bcl-2 or Bcl-xL to
protect the cells from apoptosis induced by different
stimuli (Boise et al. 1993; Minn et al. 1996).
Although Bcl-xS was one of the first pro-apoptotic
proteins to be isolated, only few things were established
concerning its mechanism of action and its role in physi-
ological cell death. However, the founding that Bcl-xS
mRNA levels were increased in some apoptotic systems,
e.g., in transient forebrain global ischemia (Dixon et al.
1997), in mammary epithelial cells (Heermeier et al. 1996)
and in injured carotid artery (Igase et al. 1999), suggested
that Bcl-xS could participate in the apoptotic pathway. In
addition, Shimizu et al. (1999) have shown that Bcl-xL
closed the mitochondrial porin channel and thus regulated
the release of cytochrome c that led to apoptosis. In gen-
eral, little is known about this gene family in mollusca and
Bcl-2 gene has only been isolated from the oyster Cras-
sostrea gigas (accession number EU678310.1).
2031
Benthic organisms and especially bivalves are widely
used as sentinel species for coastal environments moni-
toring (De Luca-Abbott et al. 2005; Gorinstein et al. 2005;
Santovito et al. 2005). Among them, sessile organisms such
as the mussel Mytilus galloprovincialis, are ideal candi-
dates for this purpose (Sole et al. 1994). These filter-
feeding organisms are very desirable bioindicator species
as they can bioaccumulate large amount of chemical pol-
lutants (Viarengo and Canesi 1991; Cajaraville et al. 2000;
Hamer et al. 2008; Petrovic et al. 2004; Jaksic et al. 2005;
Châtel et al. 2010).
Laboratory researches are so focused on the character-
ization of the cell signaling cascade in marine invertebrates
and aimed at considering it as potential biomarker for water
quality monitoring. Our previous study demonstrated that
the MAPK proteins were activated after mussel exposure to
H2O2, TBT and water soluble fraction of diesel oil (Châtel
et al. 2010). Those data will then be completed with the
present study, focused on one aspect of cellular response
after MAP kinases activation: apoptosis.
Materials and methods
Organism and investigated area
Mussels Mytilus galloprovincialis, Lamarck, 1819 (Mol-
lusca, Bivalvia) were obtained from a mariculture facility
Riviera (Limski kanal, Northern Adriatic, Croatia). The
animals were transferred to laboratory aquaria and kept in
experimental basins with running seawater (18°C) for
10 days acclimation before the experiments.
Preparation of water soluble fraction of diesel oil
One litre of fresh Diesel oil obtained from a filling station
was mixed with 4 l of sea water (SW) and stirred slowly
for 24 h, in order to enhance the dissolution of the water
soluble components of the diesel oil. The mixture was then
let for 3 h before it was poured into separating funnels and
allowed to stand overnight, in order to obtain a clean oil–
water interphase. The bottom layer, the DOWSF, was
decanted into containers (Afolabi et al. 1985) and diluted
with SW to achieved 30, 15, 7.5 and 3.25% solutions.
Mussel treatments
Mussels were exposed to tributyltin (3, 10, 30, 100 mM)
(T50202, Sigma aldrich, Deisenhofen, Germany), hydro-
gen peroxide (0.074, 0.222, 0.666, 2.000 mM) 30% (w/w)
(H1009, Sigma aldrich, Deisenhofen, Germany), and to
WSF of diesel fuel (3.25, 7.5, 15, 30%) for 1 h using 1 l of
SW and aeration, followed by recovery in clean sea water
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 Author's personal copy
2032
for 6 or 24 h. After incubation and recovery periods,
mussels (n = 3) were dissected and both gills were taken,
frozen in liquid nitrogen and stored at -80°C.
Preparation of mussels total protein extract
Mussel’s gills were homogenized with lysis-buffer (19
TBS [Tris-buffered saline], pH 7.5, 1 mM EDTA [Ethyl-
ene Diamine Tetra-acetic Acid], 1% Nonidet-P40, 10 mM
NaF [sodium fluoride], protease inhibitor cocktail [1 tablet/
10 ml] (Complete Mini, ROCHE) and 1 mM sodium
orthovanadate. After homogenization in a mortar, the clear
supernatant was obtained by centrifugation (13,0009g/
10 min/4°C) and stored at -20°C until analysis. Protein
concentrations were determined according to Bradford
(1976) using bovine serum albumin as a standard.
Determination of the Bcl-x level by Western blot
Protein extracts were mixed with sample buffer (0.5 M
Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 4% 2-mercap-
toethanol, 0.05% bromophenol blue) 1:3 and boiled for
5 min at 95°C. Proteins (20 lg per lane) were separated
using a 12% polyacrylamide gel. Western blot analysis was
performed as previously described (Böhm et al. 2000).
After transfer, membranes were incubated with a rabbit
polyclonal antibody anti-human Bcl-x (556361, BD
Pharmingen), which recognize both forms Bcl-xL and Bcl-
xS, diluted 1:5000 in blocking buffer (Roche). After
washing (3 9 5 min), membranes were incubated with
goat anti-rabbit IgG antibody coupled with alkaline
phosphatase (A9811—Sigma Aldrich) diluted 1:10000 in
blocking solution. Detection of immunocomplexes was
carried out by colorimetric reaction by using a solution of
5-bromo-4-chloro-3-indolyl phosphatase (BCIP) and
nitroblue tetrazolium (NBT). After visualization, mem-
branes were scanned and band intensity, which is the rel-
ative amount of protein expression, was quantified as
Relative Optical Densities (ROD; pixels/mm2) using the
program Odyssey ver. 1.2. Results of Bcl-xS were nor-
malized according to Bcl-xL expression.
DNA integrity: fast micromethodÒ
DNA integrity in mussel gill homogenates was measured
by the Fast MicromethodÒ. The technique may be used for
DNA integrity estimation in different types of cells and
tissues and showed to be simple, fast and sensitive (Bihari
et al. 2002). It is based on the ability of fluorochrome
(PicoGreenÒ) to interact preferentially with dsDNA in the
presence of ssDNA, RNA, and proteins even at wide range
of pH values and high ionic strengths. Briefly, to 25 ll gills
homogenate, 25 ll of lysing solution (4.5 M Urea, 0.1%
123
A. Châtel et al.
SDS, 0.2 M EDTA, pH 10) supplemented with PicoGreen
(P7581—Interchim) was added in microplates and kept in
the dark at room temperature for 30 min. DNA denatur-
ation starts after addition of 250 ll of the NaOH-EDTA
solution adjusted to achieve final pH 11.5. The extent of
DNA denaturation was followed directly in the microplate
by Fluoroscan II reader (Labsystems, Finland) measuring
the dsDNA-PicoGreenÒ complex fluorescence decay at
room temperature for 20 min. The results, expressed as
strand scission factors (SSF), were calculated as the log10
of the percentage ratio of dsDNA from treated and control
samples, respectively, after 5 min of denaturation (Jakšić
et al. 2005). For practical reasons, SSF in graphical pre-
sentations were multiplied by -1.
Caspase-Glo 3/7 assay
Caspase 3/7 activity in mussel gills was measured using the
Caspase-GloÒ 3/7 assay kit (Promega) according to man-
ufacturer instructions, which specificity on Invertebrates
has already been tested (Li et al. 2007). Briefly, the pro-
luminescent substrate containing the tetrapeptide sequence
DEVD, is cleaved by caspase 3, which results in the release
of aminoluciferin (a substrate for luciferase). After reaction
with the luciferase, luminescent signal is produced and
measured using a luminometer apparatus.
TUNEL: terminal deoxynucleotidyl transferase (TdT)
Mussel gills were fixed in isopentane frozen in liquid
nitrogen before performing 8 lm cryo-sections. Samples
were then fixed in 4% formaldehyde for 20 min at room
temperature. Each section was then washed with PBS
(phosphate buffered saline) and incubated with permeabi-
lization solution (0.1% Triton X-100 in 0.1% sodium
citrate) for 2 min on ice. Following PBS washes, the pro-
cedure was realized according to In situ cell death detec-
tion kit, Fluorescein (ROCHE), already tested in the mussel
M. galloprovincialis (Micic et al. 2001). Briefly, sections
were incubated with the TUNEL mixture (terminal
deoxynucleotidyl transferase (TdT) from calf thymus and
fluorescein-dUTP) for 1 h at 37°C. After washes in PBS,
sections were mounted and analyzed under a fluorescent
microscope.
Statistical analysis
The results of the mussel exposure experiments are given
as mean values ± S.D. of nine values (3 mussels per
condition not pooled and 3 repetitions of each test). The
measured values were compared among different groups
(model agents’ concentration) using an Analysis of Vari-
ance (ANOVA) followed by a Tukey post hoc test.
 Author's personal copy
Induction of apoptosis in mussel Mytilus galloprovincialis 2033

Statistical significance was accepted at a P \ 0.05
throughout (*), P \ 0.01 throughout (**) and P \ 0.001
throughout (***).
Results
Effect of H2O2
Bcl-xS expression
Bcl-x expression has been investigated by Western blot
using an anti-human Bcl-x antibody which recognized both
forms Bcl-xS and Bcl-xL. Our results showed that, in the
mussel M. galloprovincialis, Bcl-xL appeared as a 37 kDa
molecular weight protein (vs. 25–29 KDa in mammalians)
(Lindenboim et al. 2000) and was expressed predomi-
nantly. Moreover, an additional band of 30 kDa, cross-
reactive with the anti-human polyclonal antibody, was also
detected in the H2O2-treated mussel lysates. This additional
band was as well present in the lysates of control mussels
but in a less intensive manner (Fig. 1a). This second band
could correspond to the Bcl-xS form, an apoptotic protein
activated by mussels exposed to H2O2. However, in
mammalians, Bcl-xS molecular weight was 19.5 kDa, as
observed in the rat extract.
A 0.222 mM
Control
It was noticed that Bcl-xS expression depends upon the
concentration and the time used. Indeed, after 1 h expo-
sure, a dose dependent expression was observed for all
concentrations, except for 0.222 mM time point. Surpris-
ingly, only 0.222 mM of H2O2 presented the best recovery
of the animals, as indicated by the decrease in Bcl-xS
expression 6 and 24 h post recovery (Fig. 1b). For con-
centrations ranging from 0.666 to 2 mM, results depicted
an induction of Bcl-xS expression after 1 h exposure, a
diminution after 6 h of recovery, and then a reactivation
after 24 h.
DNA integrity
DNA integrity data of mussels exposed to H2O2 are pre-
sented at Fig. 2. Results showed that after 1 h exposure, all
H2O2 concentrations enhanced DNA damage, character-
ized by high levels in SSFx(-1) values, with maximal
increase following 1 h exposure to 0.222 mM H2O2. After
6 and 24 h of recovery in SW, mussels displayed a
decrease in DNA damage, indicating a DNA repair process.
Concerning the dose of 0.074 mM H2O2, the DNA damage
decreased appeared only after 24 h of recovery since 6 h
later, it showed a further increase in SSFx(-1) values. In
addition, it could also be noticed that the DNA repair was
more efficient after exposure to 0.074 and 0.222 mM H2O2,
compared to the highest dose used accordingly. Moreover,
Positive
negative values appeared in the SSFs of some treatments
control because DNA integrity was higher than in controls.
H2O2 – 1h (rat)

Caspase 3/7 activity

Results depicted an induction of caspase 3/7 activity after

37 KDa Bcl-xL mussel treatment with all concentrations of H2O2 but not at
Bcl-xS Bcl-xL
the same time. High induction was observed after exposure
25 KDa
Bcl-xS to 0.074 mM at 1, 6 and 24 h, while a concentration of
0.222 mM enhanced caspase 3 activity at 6 h only, as
shown in Fig. 3. Concerning 0.666 and 2 mM doses,

B H2O2
0,6 *** 1 hour exposition
Bcl-xS relative optical density

***
120 6 hours recovery
(% compared to Bcl-xL)

1h exposition 24 hours recovery

100 6h recovery ** 0,4 *
***
*
SSF×(-1)

80
24h recovery
** ** *** **
0,2
60

40 0,0
20

0 -0,2
0 0,074 0,22 0,67 2
control 0.074 mM 0.222 mM 0.666 mM 2 mM
H2O2 (mM)
Fig. 1 a Bcl-L/S protein expression in the mussel control and
exposed to H2O2 0.222 mM for 1 h. b Expression of Bcl-xS after Fig. 2 The negative DNA integrity SSF 9 (-1) values measured in
mussel exposure with different concentrations of H2O2, analysed by gill homogenates of mussels exposed to hydrogen peroxide, compared
Western blot using an anti-human Bcl-x antibody (n = 3) to mussel control (n = 9)

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2034 A. Châtel et al.

1h exposition
TBT 6h recovery

BclxS relative optical density

1400 24h recovery

(% compared to BclxL)
*** *********
1200 ***
1000 *** ***
800
600 * **
400
200 ***
***
0
control 3 mM 10 mM 30 mM 100 mM

Fig. 3 Caspase 3/7 activity in the mussel M. galloprovincialis Fig. 4 Expression of Bcl-xS after mussel exposure with different
exposed to various concentrations of H2O2 for 1 h and after 6 h concentrations of tributyltin, analysed by Western blot using an anti-
and 24 h of recovery in SW (n = 9) human Bcl-x antibody (n = 3)

maximal activity of caspase 3 was detectable after 1 h 0,6

exposure, followed by a diminution after 6 h and a reac- 1 hour exposition ***
tivation after 24 h of recovery in sea water. However, it 0,4 6 hours recovery

could be noticed that for those doses, activity levels were 24 hours recovery

SSF×(-1)
*
lower than for the other concentrations. 0,2

DNA: immunohistochemical in situ labelling (TUNEL) 0,0

In order to relate cellular apoptosis to histological locali- -0,2

0 3 10 30 100
zation, TUNEL technique has been used on H2O2 treated TBT mM
mussels. For this substance, the condition that gave high
DNA fragmentation by Fast micromethodÒ was analyzed; Fig. 5 The negative DNA integrity SSF 9 (-1) values measured in
as it was interesting to see if this DNA fragmentation was gill homogenates of mussels exposed to tributyltin chloride, compared
to mussel control (n = 9)
due to apoptosis. Transverse section across control gill
showed that it was composed of a lot of individual filaments,
containing a single layer of epithelial cells (Figs. 1a, b, 10).
The first evidence while observing H2O2-treated sections TBT. For the same doses, SSFx(-1) values tended to
was that this substance completely destroyed the gills decrease after 6 and 24 h of recovery in SW. Concerning
structure after 1 h exposure to 0.222 mM (Figs. 2a, b, 10). the exposure to 10 mM, it only induced DNA damage at
Moreover, rounded or oval labelled bodies, with varying size 6 h which was reversed at 24 h.
characteristic of apoptosis (apoptotic bodies), could be seen
(Figs. 2c, d, 10). Caspase 3/7 activity

Effect of TBT Study of caspase 3/7 activity showed that 3 and 10 mM

Bcl-xS expression
As presented in Figs. 3, 4, and 10 mM of TBT induced the
highest up-regulation of Bcl-xS, indicating apoptotic pro-
cess and its expression remained high after 6 and 24 h of
recovery. Concerning incubation with 30 mM, it only
enhanced Bcl-xS expression after 1 h exposure, since after
6 and 24 h, a reduction was observed. On the contrary,
100 mM elicited a slight increase in Bcl-xS expression.
DNA integrity
As presented in Fig. 5, dose response DNA damage was
observed after 1 h mussel exposure to 3, 30 and 100 mM of
TBT treatment enhanced a significant induction of caspase
3 activity after 24 h of recovery following exposure.
Incubation with 30 mM induced a progressive increase of
the enzyme activity at 1 h exposure, reaching a maximum
after 6 h of recovery, followed by a decrease at 24 h. A
slight activation of caspase 3 was noticed after exposure to
100 mM of TBT for 1 and 6 h time points only (Fig. 6).
DNA: immunohistochemical in situ labelling (TUNEL)
Histological analysis also showed that 1 h exposure to
100 mM TBT enhanced a disappearance of gill structure
(Figs. 3a, b, 10). This chemical induced formation of DNA
adducts, according to the strong DNA staining obtained
(Figs. 3c, d, 10).

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Induction of apoptosis in mussel Mytilus galloprovincialis 2035

TBT 0,6
***
1200
1h exposition *** 1 hour exposition
Luminescence (RLU)

1000 6h recovery 6 hours recovery *** ***

24h recovery
0,4

SSF×(-1)
24 hours recovery
800
***
*** *** *** * ***
600 *
400
* 0,2

200

0 -0,1
control 3 mM 10 mM 30 mM 100 mM 0 3,75 7,5 15 30
Diesel Oil (%)
Fig. 6 Caspase 3/7 activity in the mussel M. galloprovincialis
exposed to various concentrations of TBT for 1 h and after 6 h and Fig. 8 The negative DNA integrity SSF 9 (-1) values measured in
24 h of recovery in SW (n = 9) gill homogenates of mussels exposed to water soluble fraction of
diesel oil, compared to mussel control (n = 9)

Effect of WSF of diesel oil Caspase 3/7 activity

Bcl-xS expression While observing caspase 3/7 activity, it could be noticed

Following diesel oil exposure, Bcl-xS expression was
enhanced dose-dependently at 1 h from 7.5 to 30%. Sur-
prisingly, at 7.5%, Bcl-xS levels were closed to control
levels. For 3.25 and 15%, an increase in Bcl-xS expression
was enhanced at 1 h, followed by a slight diminution in the
expression after 6 and 24 h of recovery in SW, whereas for
the two other doses (7.5 and 30%), the expression was
re-increased at 24 h (Fig. 7).
DNA integrity
Mussel exposure for 1 h to diesel oil concentration varying
from 0 to 15% resulted in a clear dose response of DNA
integrity loss. The SSF 9 (-1) values obtained after a
period of 6 h recovery time increased following 3.25 and
7.5% DOWSF exposure but decreased for the highest doses
(15 and 30%). Finally, after 24 h of recovery, mussels
displayed decline levels of SSFx(-1) values (Fig. 8).
DO
Bcl-xS relative optical density
that DOWSF induced the highest enzyme levels after 6 h
of recovery following treatment with 3.25, 7.5 and 15%.
On the contrary, after 1 h of incubation to DOWSF 30%,
the enzyme activity was already impacted (Fig. 9).
DNA: immunohistochemical in situ labelling (TUNEL)
Contrarily to the other chemicals tested, a 6 h recovery
period, post exposure to 7.5% of diesel oil, was not suffi-
cient to produce any alteration in mussel gill filament
structure (Figs. 4a, b, 10), but induced like the others,
apoptosis, as demonstrated by the highly stained DNA in
cells. Moreover, apoptotic bodies were detected in the
epithelial cell layers (Figs. 4c, d, 10).
Discussion
The aim of this study was to get a better understanding of
the cell signaling pathway leading to apoptosis in the
mussel M. galloprovincialis exposed to marine pollutants.
In doing so, results obtained in a previous study concerning
MAP kinase activation in mussels exposed to the same

1h exposition
(% compared to Bcl-xL)

6h recovery *** ***

200 24h recovery
160
***
*** ***
120 * ** **
80

40

0
control 3.25 % 7.5 % 15 % 30 %

Fig. 7 Expression of Bcl-xS after mussel exposure with different Fig. 9 Caspase 3/7 activity in the mussel M. galloprovincialis
concentrations of water soluble fraction of diesel oil, analysed by exposed to various concentrations of WSF of diesel oil for 1 h and
Western blot using an anti-human Bcl-x antibody (n = 3) after 6 h and 24 h of recovery in SW (n = 9)

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2036
Fig. 10 TUNEL stained transverse section across adjacent gill filaments of Mytilus galloprovincialis control (1), H2O2-treated (2), TBT-treated
(3), diesel-treated (4), a and b (910), c and d (9100)
conditions (Châtel et al. 2010) were compared to the
present results based on apoptosis.
Effect of H2O2
H2O2 is a known genotoxic agent and due to its marked
effectiveness in inducing DNA fragmentation, through
intracellular generation of hydroxyl radicals (Singh et al.
1988; Visvardis et al. 1997), it was used as positive control
of apoptosis induction (Singh et al. 1988; Kruszewski et al.
1994). Results clearly showed the induction of a protein of
*44 KDa after mussel exposure to any H2O2 concentra-
tions. This protein was supposed to be Bcl-xS, the form of
Bcl-x which has a pro-apoptotic role. However, Bclx has
never been characterized in the mussel M. galloprovincialis
so far, and only little were known about its mechanism of
action. But in this study, it was interesting to analyze this
protein as it gives indications concerning both apoptosis
and cell survival. Indeed, Bcl-xL and Bcl-xS are alternative
splicing forms (Boise et al. 1993) and the long form
(Bcl-xL) suppresses cell death, whereas the short form
(Bcl-xS) acts by forming dimers with Bcl-xL or Bcl-2,
neutralizing the activities of these anti-apoptotic proteins
and, thereby, promoting apoptosis (Boise et al. 1993; Fang
et al. 1994). Our data showed that 1 h exposure to hydrogen
123
A. Châtel et al.
peroxide induced an increase in Bcl-xS expression with
respect to Bcl-xL, indicating enhancement of an apoptotic
process in the mussel cells. Shou et al. (2002) also demon-
strated in rat cortical cells, that 24 h cyanide treatment
increased Bcl-xS protein expression, leading to apoptosis.
Lindenboim et al. (2000) demonstrated in PC12 cells,
that caspase activity was required for apoptosis induced
by Bcl-xS. In our study, caspase 3 activity was enhanced
after mussel exposure to H2O2 and this activity could be
correlated with Bcl-xS expression. Stridh et al. (1998)
showed in Jurkat cells exposed to H2O2, that caspase
activation occurred 1 h after cytochrome c release from
the mitochondria, which has been shown to be induced by
Bcl-xS in PC12 cells (Lindenboim et al. 2004). For all of
the doses used, induction of caspase activity and Bcl-xS
expression were both detected at 1 h, except for a
0.222 mM H2O2 concentration which triggered caspase 3
activity 6 h later than Bcl-xS expression; this could be
explained by the fact that in tissue, cells undergo apop-
tosis asynchronously.
Moreover, in a previous study, we demonstrated that
p38 and ERK MAPK were enhanced after mussel exposure
to the same conditions, which is in agreement with Zhuang
et al. (2007) who showed that only ERK was responsible of
H2O2-mediated apoptosis through caspase 3 activation in
 Author's personal copy
Induction of apoptosis in mussel Mytilus galloprovincialis
renal epithelial cells, even though p38 was also activated
with this substance.
Following the Fenton reaction, H2O2 is responsible for
direct nucleobase modification by generating OH- which
can add double bounds to DNA bases (Von Sonntag 1987;
Halliwell and Aruoma 1991). This oxidative DNA damage
takes the form of Single Stranded Break (SSBs) and to a
lesser extend Double Stranded Breaks (DSBs), alkaline
labile sites and various species of oxidized purines and
pyrimidines (Von Sonntag 1987).
So as to analyze DNA damage in mussels exposed to
H2O2, two techniques were employed, TUNEL and Fast
micromethodÒ, both are based on the detection of DNA
strand breaks. The Fast micromethodÒ assay is not specific
to apoptosis since it detects SSBs, alkaline labile sites as
well as incomplete excision repair (Jaksic and Batel 2003)
whereas the TUNEL assay relies on DNA nick character-
ization which is a hallmark of apoptosis. Results showed
that DNA damage was increased after 1 h exposure to all
H2O2 concentrations tested which was followed by a
diminution at 6 and 24 h. Moreover, at 1 h, apoptotic
bodies could be distinguished with dense chromatin, highly
fragmented, confirming the presence of apoptosis. The
decrease in DNA damage observed after 6 and 24 h of
recovery in sea water was due to the fact that cells which
were too affected by the chemical underwent apoptosis.
Liu et al. (1997) identified a DNA fragmentation factor
(or DFF), composed of DFF 40 and 45, enabled to cause
chromosomal DNA fragmentation in the presence of an
activated caspase 3. In the mussel, upon activation of
apoptosis, DFF45 could be cleaved by caspase 3 and dis-
sociate from DFF40 as demonstrated by Liu et al. (1997)
and Nagata (2000). Free DFF40 could be able to cleave
DNA into oligonucleosomal size fragments, for apoptosis
induction (Zhang and Xu 2000).
In addition, gill structure was destroyed with this
chemical, also observed by Tort et al. (2002) in rainbow
trout gills after hydrogen peroxide exposure.
All those biomarkers demonstrated the presence of
apoptosis in H2O2-treated mussels, hence validating our
experimental procedures before testing two major pollu-
tants of the marine environment.
Effect of TBT
Although present applications include molluscicides, stone
preservation and disinfectants, the most important usage of
TBT remains in wood preservatives and anti-fouling paints.
Even though, recommendations have been made by the
International Maritime Organization (IMO) to internation-
ally prohibit the use of organotin compounds as biocides in
antifouling paints by January 2008, it remains a widespread
2037
environmental contaminant (Kimbrough 1976; Tanabe
et al. 1998). Indeed, in harbour areas, TBT concentrations
varied from 100 to 1,000 ng/l (Volkman 2006). In the
present study, higher TBT concentrations were used com-
pared to field concentrations, since a short-term exposure
was realized.
Our results showed that Bcl-xS expression was
enhanced at 1 h exposure with all of TBT doses and
highest levels were obtained with 3, 10 and 30 mM.
Caspase 3 activity was also detected following mussel
exposure to this xenobiotic. However, for the two lowest
concentrations tested (3 and 10 mM), caspase 3 was acti-
vated 18 h later than MAP kinase (p38 and JNK) activation
observed in a previous study (Châtel et al. 2010). This
delay was reduced at 6 h for a 30 mM TBT concentration.
Concerning the mussel incubation with 100 mM, activation
of caspase 3 and p38 were simultaneous, suggesting that at
this dose, caspase 3 depended on p38 activation. However,
in the rainbow trout exposed to TBT, Owuor and Kong
(2002) demonstrated that caspase 3 was dependent of JNK
activation. This could also be the case for the mussel but at
low concentrations of TBT. It appears that at low TBT
doses, Bcl-xS would be immediately induced by MAP
kinases p38 and JNK, followed by caspase 3 activation
18 h later. Notwithstanding, at high concentrations, p38
alone would be responsible for the slight increase in Bcl-xS
expression and immediate activation of caspase 3. Nakatsu
et al. (2007) have also demonstrated that PC12 cells
exposed to 100 lg/l (30 mM) of TBT induced caspase 3
activity via JNK activation while 300 lg/l (100 mM)
triggered ROS production and not JNK activation. Chen
and Tan (1998) and Morishima et al. (2001) have also
correlated ROS and JNK to caspase 3 activity.
In the literature, TBT is known as a genotoxic agent. It
acts as modifying base methylation, an essential bio-
chemical process necessary for gene expression (Wang
et al. 2009). In addition, TBT triggers ROS production that
has a direct impact on DNA (Von Sonntag 1987). Fafandel
et al. (2003) observed for sponges exposed to TBT, a
modification of KRS kinase phosphorylation (a stress-
responsive protein serine/threonine kinase), protein impli-
cated in apoptosis. This data is in correlation with our
results since apoptotic DNA fragmentation was observed.
DNA breaks as well as apoptotic bodies could be
observed by the TUNEL technique in TBT-treated mussels.
Previous studies have also demonstrated that DNA frag-
mentation occurred after M. galloprovincialis exposure to
TBT, in a dose and time-dependent manner (Micic et al.
2001). Moreover, those treated-mussels displayed a disor-
ganization of gill filaments. This observation was in
accordance with the study of Micic et al. (2001) who also
noticed a destruction of gill structure in mussels injected
with 1–5 lg/g of TBT, accompanied by a disappearance of
123
 Author's personal copy
2038
interfilament junctions, cilia and changes in endothelia
cells.
The same authors suggested that dsb (double stranded
break) DNA, resulting in the degradation of higher-order
DNA or in DNA fragmentation at the oligonucleosomal
level, are the initial events induced by in vivo TBT treat-
ment leading to nuclear morphological changes.
Effect of water soluble fraction of diesel oil
Diesel fuel pollution represents the major water contami-
nation in current society. They are introduced in the
environment from a variety of sources including forest
fires, volcanic eruptions, diesel, tobacco (White et al. 1994;
Phillips and Grover 1984). For the last decade, coastal
ecosystems have been subjected to increase hydrocarbon
contamination from compounds such as polycyclic aro-
matic hydrocarbons (PAHs). As an example, PAHs con-
centration in the Adriatic Sea reaches 100 lg/l (Gambaro
et al. 2007; Bihari et al. 2007). PAH, lipophilic molecules,
are metabolized by the cytochrome P450 monoxygenase
family (CYP1) in highly reactive metabolites that induce
the formation of DNA adducts (Pelkonen and Nebert
1982).
Results showed that both caspase 3 and Bcl-xS were
activated after mussel exposure to diesel oil. For the lowest
doses (3.25, 7.5, 15%), it has been noticed that maximal
caspase 3 activity was enhanced 6 h later than Bcl-xS
expression. In addition, in a previous study, activation of
the MAP kinase p38 and JNK in mussels exposed to the
same conditions has been demonstrated (Châtel et al.
2010). Those observations are in accordance with Landvik
et al. (2007) who showed in hepathoma cells that nitro-
PAH (Nitrated-polycyclic aromatic hydrocarbons), also
present in diesel oil, induced p53 accumulation in the
nucleus, Bcl-xL down-regulation, caspase 3 activation as
well as p38 and JNK MAPK phosphorylation. However, on
the contrary to TBT, it appeared that p38 was responsible
of PAHs-mediated apoptosis according to Chen et al.
(2003) who demonstrated that a p38 inhibitor failed to
activate caspase 3 induced by benzo{a}pyrene. Rummel
et al. (1999) also showed that benzo{a}pyrene induced
apoptosis independently of JNK activation.
TUNEL analysis showed the presence of DNA breaks,
after 1 h mussel exposure with diesel oil, confirmed by the
Fast MicromethodÒ assay, with abundance of apoptotic
bodies.
Hazardous effects of PAHs arise as a result of oxidative
biotransformation producing highly DNA-reactive metab-
olites which have been recognized as carcinogenic, muta-
genic and cytotoxic compounds (Torres-Bugarin et al.
1998; Woodhead et al. 1999).
123
A. Châtel et al.
Micic et al. (2002) have demonstrated in the marine
mussel exposed to benzo{a}pyrene (5–20 lg/l) induced
DNA repair by DNA adduct excision and not by apoptosis
induction, contrarily to our conclusions.
The genotoxic potency of ten polycyclic aromatic
hydrocarbons (anthracene, 7,12-dimethylbenz[a]anthra-
cene, benz[a]anthracene, dibenz[a,h]anthracene, dibenz[a,c]
anthracene, 3-methylcholanthrene, benzo[a]pyrene, benzo
[e]pyrene, chrysene and pyrene) has been well character-
ized (Nishikawa et al. 2005). Cells with micronuclei were
found to be increased in gills and hemolymph of marine
molluscs treated with benzo(a)pyrene (Burgeot et al. 1996;
Venier et al. 1997; Siu et al. 2004), and with dimethyl-
benz(a)antracene (Bolognesi et al. 1996). Comet assay
presented clear dose- and time-dependent responses to
benzo(a)pyrene exposure in the bivalve Perna viridis (Siu
et al. 2004).
Proposed mechanism for mussel signaling pathway
induction by TBT and diesel oil
The genotoxic agents, TBT and diesel oil, induced from
1 h exposure, a DNA fragmentation as observed by both
Fast micromethodÒ and TUNEL assays and the presence of
apoptotic bodies confirmed apoptosis induction. Both
chemicals are known to induce ROS production and
especially H2O2 in the cell in vertebrate model as well as in
invertebrates (Pruski and Dixon 2002; Abele et al. 2002;
Lannig et al. 2006). The main target of ROS is the mito-
chondria, where they can enhance pro-apoptotic protein
activation like Bcl-xS (observed at 1 h). However, for
those compounds, caspase 3 activity was enhanced later for
the lower concentrations, which indicated that caspase 3
activity was not necessary for TBT and diesel-induced
apoptosis at those doses. In these conditions, it appeared
that apoptosis was mediated by a caspase 3-independent
pathway (Fig. 11). A recent study showed that the caspase
3 inhibitor fails to prevent apoptosis in the oyster Cras-
sostrea virginica following infection with a parasite
(Hughes et al. 2010), suggesting the existence of a caspase-
independent death pathway in this organism. The most
prominent caspase-independent death effector molecules
are the apoptosis-inducing factor (AIF), a phylogenetically
conserved mitochondrial flavoprotein, and endonuclease G,
a mitochondrion-specific nuclease (van Loo et al. 2001; Li
et al. 2001; Cande et al. 2002; Tait and Green 2008). Those
two proteins translocate from the mitochondria to the
nucleus where they bind to DNA, leading to DNA frag-
mentation and cell death. A partial gene sequence has been
cloned of an AIF-like gene from oyster (accession #
MGID89694).
Another protein also plays an important role in apop-
tosis signaling in molluscs, p53, cloned in Mytilus
 Author's personal copy
Induction of apoptosis in mussel Mytilus galloprovincialis
High concentrations of
TBT
PAHs
p38 / JNK ROS
DNA damage Bcl-xS
(TUNEL)
Caspase
APOPTOSIS 3 impact of other chemicals as well as environmental
p38 / JNK ROS
Low concentrations of
TBT
PAHs
Fig. 11 Hypothetical cell signaling cascade leading to apoptosis in
the mussel, activated with various concentrations of TBT and diesel
oil
(Banni et al. 2009), which mediates apoptosis either via
translocation into the nucleus where it up regulates pro-
apoptotic gene transcription or by translocation to the mito-
chondria where it inactivates anti-apoptotic proteins of the
Bcl-2 family (Böttger et al. 2008), modulating the expression
of Bcl-2, Bcl-xL and Bcl-xS (Chiu et al. 2003), perhaps
explaining in our conditions the increase of Bcl-xS at 1 h.
On the contrary, high doses of TBT and diesel as well as
all doses of H2O2 induced caspase 3 activity, Bcl-xS
expression and DNA fragmentation simultaneously after
1 h exposure, showing that in those conditions, apoptosis
was caspase 3-dependent. The effects of 2 lM TBT
(equivalent to 300 lg/l) on PC12 cells have been shown to
be mediated via excessive ROS production, contrarily to
lower dose which was mediated via MAPK signaling
pathway (Nakatsu et al. 2007). Highest levels of ROS at
high concentrations of TBT and diesel, as well as extra-
cellular ROS (H2O2) itself, could have a faster impact on
the mitochondria and thus explain the detection of caspase
3 activity at 1 h.
Conclusive remark—perspectives
In this preliminary study, we showed that H2O2, TBT and
DOWSF induced differential activation of the MAPK that
lead to different apoptotic signaling pathway activation
depending on the concentration tested. For the first time,
Bcl-xS expression was studied in the mussel and we
showed that this protein was necessary for apoptosis
induction in the mussel. In addition, we demonstrated
that two apoptotic pathways were present in the mussel
M. galloprovincialis, dependent or not on caspase 3, which
2039
activation varied with the nature of the xenobiotic and/or
its concentration.
To conclude, the overall study could provide additional
response elements concerning the cell signaling pathway in
the mussel but above all that, could open some future
perspectives: (1) to use specific inhibitors of caspase 3 and
MAP kinase so as to understand the interaction between
those proteins; (2) to characterize the gene encoding Bcl-x
in M. galloprovincialis so as to produce specific antibody;
(3) to perform longer exposure studies to chemicals so as to
mimic reality field conditions and (4) to evaluate the
parameters such as temperature, salinity in the objective, in
fine, to envisage this signaling pathway as biomarker in
biomonitoring studies.
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