Aquatic Toxicology 99 (2010) 7385

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Apoptosis and necroptosis are induced in rainbow trout cell lines

exposed to cadmium
Gerhard Krumschnabel a, , Hannes L. Ebner b , Michael W. Hess b , Andreas Villunger a
a
b

Division of Developmental Immunology, Biocenter, Medical University Innsbruck, Fritz-Preglstr. 3, Innsbruck, Austria
Division of Histology and Embryology, Medical University Innsbruck, Innsbruck, Austria

a r t i c l e

i n f o

Article history:
Received 17 February 2010
Received in revised form 30 March 2010
Accepted 2 April 2010
Keywords:
Cadmium
Copper
Apoptosis
Necroptosis
Gill
Liver

a b s t r a c t
Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated
apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the
process of cadmium-mediated toxicity, but the exact mechanism remains ill-dened and may depend on
the affected cell type. This study investigated which mode of cell death may be responsible for cell death
induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to
inhibitors of necroptosis or apoptosis, respectively. It was observed that intermediate levels of cadmium
that killed approximately 50% of the cells over 96120 h of exposure caused cell death that morphologically resembled apoptosis and was associated with an increase of apoptotic markers such as the number
of cells with diminished DNA content (sub-G1 cells), condensed or fragmented nuclei, and elevation of
caspase-3 activity. At the same time, however, cells also lost plasma membrane integrity, as indicated
by uptake of propidium iodide, showed a decrease of ATP levels and mitochondrial membrane potential,
and displayed cell swelling, signs associated with secondary necrosis, or equally possible, necroptotic cell
death. Importantly, many of these alterations were at least partly inhibited by the necroptosis inhibitor
necrostatin-1 and were to a lesser extent also sensitive to the pan-caspase inhibitor zVAD-fmk, indicating
that multiple modes of cell death are concurrently induced in cadmium-exposed trout cells, including
necroptosis and apoptosis. Cell death appeared to lack concurrent radical formation, consistent with
genetically regulated necroptotic cell death, but was characterized by the rapid induction of DNA damage
markers, and the early onset of disintegration of the Golgi complex. Comparative experiments evaluating copper-toxicity indicated that in comparison to cadmium much higher concentrations of this metal
were required to induce cell death and that neither necrostatin-1 nor a pan-caspase inhibitor conferred
protection, suggesting that additional modes of cell death can be triggered in response to poisoning with
heavy metals.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Cadmium (Cd) is an important toxicant both in terrestrial and
aquatic environments. Depending on the cell type and concentration, it may induce cell death via apoptosis, characterized by
typical features such as caspase activation and DNA fragmentation
(Habeebu et al., 1998; Pulido and Parrish, 2003), or necrosis typied
by ATP depletion and plasma membrane permeabilization (Lopez
et al., 2003; Yang et al., 2007). Furthermore, in some instances
a less clearly dened mode of cell death was described involving both apoptotic and necrotic features (Lee et al., 2006; Sancho
et al., 2006; Shih et al., 2003). Recently, a mode of programmed
necrosis, termed necroptosis has been identied, in which cell
death occurs with largely necrosis-like morphological alterations,

Corresponding author.
E-mail address: Gerhard.Krumschnabel@i-med.ac.at (G. Krumschnabel).
0166-445X/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2010.04.005

but following apparently well-dened molecular signaling pathways that recruit components of the extrinsic as well as intrinsic
apoptotic cell death machinery (Christofferson and Yuan, 2010). A
crucial component of this pathway is the serine/threonine kinase
receptor-interacting protein-1 (RIP-1), the pharmacological inhibition of which by the small molecule inhibitor necrostatin-1 (Nec-1)
was in fact key to identifying necroptosis (Degterev et al., 2005).
A closer characterization of necroptosis was achieved by use of
the mouse brosarcoma cell line L929 in which addition of the
physiological ligand tumor necrosis factor- (TNF), or inhibition
of caspases using zVAD-fmk induces cell death that is sensitive
to Nec-1 inhibition (Degterev et al., 2005). In addition to such a
receptor-mediated, extrinsic stimulation, Nec-1 sensitive cell death
involving RIP-1 may also be triggered through intrinsic clues, in
particular in response to severe DNA damage (Festjens et al., 2006;
Xu et al., 2006; Zong et al., 2004). In line with this, it was recently
shown that Cd, which may induce DNA damage as well (Bertin and
Averbeck, 2006; Joseph, 2009; Viau et al., 2008), induces necropto-

74

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

sis in Chinese hamster ovary cells (Hsu et al., 2009). Specically, it

was reported that Nec-1 did not prevent the elevation of intracellular calcium levels and reactive oxygen species (ROS) associated
with Cd-exposure, whereas it attenuated Cd-induced reduction of
mitochondrial membrane potential and plasma membrane permeabilization. This suggests that calcium and ROS act upstream of
RIP-1, while mitochondrial alterations and ultimately the breakdown of cell integrity are downstream of the kinase. In addition,
Nec-1 caused signicantly enhanced DNA binding activity of the
pro-survival transcription factor NFB, which might be taken to
indicate that Nec-1 may not only protect from Cd-induced toxicity
by inhibiting RIP-1 but also ameliorate DNA damaging effects by
facilitating transcription of pro-survival genes thereby enhancing
damage repair and subsequent cell death signaling.
Similar to the ndings reported for mammalian cell systems,
various studies with teleost cells indicated apoptotic and necrotic
death upon Cd-exposure (Lyons-Alcantara et al., 1998; Risso-de
Faverney et al., 2001; Xiang and Shao, 2003). So far, however, the
possible occurrence of necroptotic cell death in response to Cd has
not been addressed in cells from teleosts. Thus, despite the fact
that apoptosis signaling appears largely conserved among sh and
mammals (Krumschnabel and Podrabsky, 2009), it has not yet been
shown if this also holds for necroptosis. The present study was
therefore conducted to elucidate if Cd-exposure of teleost cell lines
derived from trout elicits cell death that is sensitive to inhibition
by Nec-1, and to characterize the physiological alterations associated with this. Furthermore, although such an in vitro study does
not reect a truly environmentally relevant situation, since sh are
frequently exposed to this pollutant we also wanted to gain information regarding the in vivo toxicity of Cd in sh. We therefore
used both a gill cell line, RTgill-W1, representing the cell type that
is usually at rst and most directly exposed to environmental pollutants, and a liver cell line, RTH-149, representing those cells where
many toxicants including metals may be accumulated and detoxied. The occurrence of specic cell death features in either cell type
may thus provide information pertaining to their use as environmental markers of, e.g. the effects of acute versus chronic exposure
to Cd and/or information related to concentrationresponse relations characteristic for each cell type. For reasons of comparison,
we also examined what type of cell death is induced by copper, a
second relevant pollutant, in these trout cells, since previous studies on primary trout hepatocytes indicated mixed apoptotic and
necrotic death but did not address the possibility of necroptosis
(Krumschnabel et al., 2005; Nawaz et al., 2006).

2. Materials and methods

2.1. Chemicals and antibodies
Leibovitz L-15 medium and fetal bovine serum (FBS)
were from Gibco (Invitrogen, Vienna), l-glutamin, penicillin/streptomycin and trypsin/EDTA were from Cambrex
Bioscience (Oberhaching, Germany). Fluorescence indicators
dichlorouorescein diacetate (DCF-DA), 5,5V,6,6V-tetrachloro1,1V,3,3V-tetraethylbenzimidazolylcarbocyanine iodide (JC-1),
Hoechst 33342, and tetramethylrhodamine methylester (TMRM)
were obtained from Molecular Probes (Leiden, The Netherlands). CellTiter-Glo and the Apo-ONE homogeneous caspase-3/7
assay kit were from Promega (Mannheim, Germany), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
from Roche Diagnostics (Vienna, Austria), the poly-(ADPribose) polymerase inhibitor 3-aminobenzamide, propidium
iodide (PI) and 4 ,6-diamidino-2-phenylindole (DAPI) were
from Sigma (Deisenhofen, Germany). Necrostatin-1 (Nec-1)
and the hsp-90 inhibitor 17-(dimethylaminoethylamino)-17-

demethoxygeldanamycin (17-DMAG) were from Eubio (Vienna,

Austria), the pan-caspase inhibitor Z-Val-Ala-dl-Asp(OMe)uoromethylketone (zVAD-fmk) from Bachem (Weil am Rhein,
Germany), cyclosporine A (CsA) from LC Laboratories (Woburn,
MA, USA). Inhibitors of mitogen-activated protein kinases U0126
and SB203580 were obtained from Calbiochem (Merck, Darmstadt,
Germany), SP600125 from A.G. Scientic Inc. (Munich, Germany).
N-(2-Quinolyl)valyl-aspartyl-(2,6-diuorophenosy)methyl ketone
(QVD) was from Alexis Biochemicals (Lausen, Switzerland).
Antibodies against H2AX (#9718), phospho-ATM (#4526),
phospho-Chk-2 (#2661) were purchased from New England
Biolabs (Frankfurt am Main, Germany), FITC-labeled mouse antiGM-130 was from BD Transduction Laboratories (Vienna, Austria),
and secondary antibodies Alexa Fluor 488 goat anti-rabbit and
Alexa Fluor 546 goat anti-mouse from Invitrogen (Vienna, Austria).
Cd and Cu were applied from stock solutions of CdCl2 and CuCl2
dissolved in distilled water.
2.2. Cell lines and cell culture
Rainbow trout-derived cell lines, the gill cell line RTgill-W1 and
the hepatoma line RTH-149, were obtained from ATCC (Manassas,
VA, USA) and were cultured in Leibovitz L-15 medium with 10%
FBS, 2 mM l-glutamine and 1% penicillinstreptomycin at 21 C in
an air atmosphere. For experimental exposures, which were also
all conducted at 21 C in air atmosphere, serum concentration was
reduced to 0.5%, which was found adequate to still support cell
growth but to reduce potential binding of metals.
2.3. Experimental exposures
Cells were initially exposed to concentrations between 10 and
500 M Cd and Cu over a time period ranging from a few hours
to 5 days. Having established metal concentrations and exposure
times leading to intermediate levels of cell death, a number of
different inhibitors of signaling molecules involved in classical
apoptosis or in necroptosis was applied and the impact of these
inhibitors on viability and cell death-related parameters determined in metal exposed cells was evaluated. Concentrations of
metals and inhibitors as well as specic time points are as detailed
in Section 3 for each experiment.
2.4. Cell viability and caspase activity measurements
Cell viability was determined using the MTT kit from Roche as
instructed by the suppliers. In brief, cells were seeded into 96-well
plates at a density of 15,000/well (RTgill-W1) or 7500/well (RTH149) and incubated at 21 C overnight prior to experimentation.
Then cells were once washed with low-serum medium and subsequently exposed to the desired treatment as indicated in Section
3. If specic inhibitors were used, a 1 h pre-incubation period preceded experimental exposures. At the end of the incubation period
MTT was added and the cells left to metabolize the tetrazolium salt
for 4 h. Following addition of solubilization solution and incubation
for >6 h, absorbance was measured on a multi-well plate reader at
595/620 nm.
For the determination of caspase activity, cells were cultured in
the same way and at the end of experimental treatments caspase3/7 assay mix was added to each well, the reaction incubated for
30 min and uorescence was then measured at 485 nm excitation
and 538 nm emission. Basically the same holds for measurements
of ATP contents, for which after treatments the CellTiter-Glo assay
mix was added to wells and after 30 min of incubation luminescence values were determined in a multi-well plate reader with
luminescence detection.

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

A marker of apoptotic cells is the occurrence of diminished

DNA content, as assessed by the sub-G1 assay (Nicoletti et al.,
1991). For this purpose cells were cultured in 6 cm cell dishes and
exposed to treatments before cells were collected by trypsinization (including oating cells), pelleted by centrifugation and then
xed in 70% ice-cold ethanol. After one wash with PBS cells
were RNAse digested for 20 min at 37 C and then incubated with
50 g/ml PI for 10 min before DNA contents were analyzed with a
FACScalibur ow cytometer (BD Biosciences, Erembodegem, Belgium).
For the determination of plasma membrane permeabilization
as occurs in necrosis, cells were cultured and treated in 24-well
plates, and subsequently co-incubated with 5 g/ml of the membrane impermeable dye PI, which only enters dead cells, and
2 g/ml Hoechst 33342, a nuclear stain permeating viable and
dead cells. Following 5 min of incubation, cells were photographed
under a uorescence microscope using excitation/emission lters specic for Hoechst 33342 and PI and the number of
total and PI-positive cells were analyzed using Image J software.
2.5. Immunouorescence stainings
Cells were seeded onto sterilized glass cover slips and left to
rmly attach for at least 24 h. The cultures were then exposed to
the desired treatment, briey washed with PBS, and then xed for
15 min at room temperature with 4% PFA (in PBS). Following three
washes with PBS cells were incubated for 1 h with PBS containing 0.1% Triton X-100, 1% bovine serum albumin and 10% fetal calf
serum for permeabilization and blocking. Primary antibodies were
diluted 1:50 (anti-GM-130) or 1:100 (all others) in blocking solution and samples incubated overnight at 4 C. After three further
washes with PBS, cells were incubated with secondary antibody
(1:100 in blocking solution) for 1 h at room temperature, washed
with PBS, incubated with DAPI (2 g/ml, in PBS) for nuclear staining for 10 min at room temperature and washed one more nal
time. Cells were then xed on microscope slides with Vectashield
antifade mounting medium (Vector Laboratories Burlingame, CA).
Cell images were collected with a Leica SP5 confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) using a
63 glycerol immersion objective, with acquisition through the LAS
AF acquisition software Version 2.1.0, applying three averages on
each channel, a resolution of 1024 1024 pixels, and pinholes set
to 1 Airy unit. Post-acquisition image processing, i.e. background
correction, adjustment of brightness and contrast and export to
tif-format, were done with Image J software.

75

2.7. Production of reactive oxygen species (ROS)

The formation of reactive oxygen species was examined in cells
seeded and exposed in 96-well plates as described for the MTT
viability assay (Section 2.4). After experimental exposure well contents were aspired, 50 l PBS containing 5 M of the ROS indicator
DCF-DA were added and cells incubated in the dark for 30 min
before measuring uorescence at 485 nm excitation and 538 nm
emission.
2.8. Electron microscopy
For transmission EM samples were subjected to rapid cryoimmobilisation by means of high-pressure freezing instead of
chemical xation, which greatly improves the preservation of ultrastructural features. In brief, cells were cultured on carbon-coated
sapphire coverslips, stimulated as required and subjected to highpressure freezing followed by freeze-substitution and epoxy resin
embedding (Hess et al., 2000). Sections were analyzed with a
Philips CM120 EM (F.E.I., Eindhoven, Netherlands) and images were
recorded with a MORADA digital camera (SIS, Mnster, Germany).
Contrast and brightness of the digital images were optimized by
using grey scale modication and high-pass ltering of the ADOBE
photoshop software (Version 9).
2.9. Time-lapse imaging
For live cell imaging cells were cultured in 12-well plates, chemicals added as appropriate, the plates sealed with Paralm to prevent
evaporation and mounted on the stage of a Cell-IQ real-time image
capture system (Chip-Man Technologies Ltd, Tampere, Finland).
Images were then captured at 30 min or 1 h intervals and timestamped and exported as movie les by use of Image J software.
2.10. Statistics
Data shown are means SE of n cultures. All experiments were
conducted on at least 3 different sub-cultures individually maintained before the experiments. In addition, all experiments were
repeated at least twice on cells from different passage numbers.
Statistical differences were evaluated by ANOVA followed by the
appropriate post hoc tests, with a p value <0.05 considered as signicant.
3. Results
3.1. Concentrationresponse relationships of metals and cell
viability

2.6. Quantication of mitochondrial membrane potential (MMP)

Alterations of MMP were determined with JC-1 loaded cells
using FACS analysis. In brief, cells were cultured in 6-well plates,
exposed to stimuli, trypsinized and then incubated for 30 min in
the dark with 4 M JC-1 dissolved in Leibovitz medium. Cells were
then spun down in Eppendorf tubes, washed once with PBS and
re-suspended in PBS. FACS measurements were done on a FACScalibur ow cytometer determining red (cells with high mitochondrial
potential) and green (cells with low mitochondrial potential) uorescence.
As an alternative method for the quantication of MMP cells
were cultured in 96-well plates as above (Section 2.4), treated as
desired and then incubated with 200 nM TMRM for 30 min. Subsequently the wells were once washed with PBS before re-addition
of saline and then uorescence emitted from TMRM retained in
energized mitochondria was determined at 544 nm excitation and
590 nm emission.

A rst series of experiments investigated the concentration

response relation between the concentrations of Cd or Cu and cell
viability. Pilot experiments had shown that rather high concentrations of Cd (>200 M) were required to induce signicant cell
death within 24 h and this was then almost exclusively necrotic
(not shown). Thus, lower concentrations and prolonged exposure
times were chosen and it was observed that after 120 h of incubation a gradual concentration-dependent decline in cell viability
occurred in RTgill-W1 cells, with about 50% cell death determined
in the presence of 50 M Cd (Fig. 1A). RTH-149 hepatoma cells were
somewhat more sensitive and showed nearly 60% cell death after
96 h of exposure to 50 M Cd (Fig. 1B). Cu appeared to be less toxic
to both cell lines and in RTgill-W1 cells we even observed a slight
proliferative effect of Cu up to concentrations of 100 M (Fig. 1A).
Above this level, however, Cu reduced cell viability in both cell lines
and at 1 mM it killed 85% and 90% of all gill and hepatoma cells,
respectively (Fig. 1A and B).

76

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Fig. 1. Concentration-dependent cell death of trout cells exposed to Cd or Cu. Cell viability, as estimated from MTT conversion, of RTgill-W1 cells exposed to Cd or Cu at the
indicated concentration for 120 h (A) and of RTH-149 cells exposed for 96 h (B). Panel (C) shows examples of FACS analyses of gill cells used to determine the percentage
of cells with diminished DNA content and in (D) the quantitative relation between the concentration of Cd and Cu and the number of sub-G1 cells is depicted. Data are
means SE of 3 (Cd in RTgill-W1 and panel D) and 47 cultures used in at least 2 independent experiments. N.d.: not determined.

Concentration-dependent changes elicited by the metals were

also established for the number of cells with diminished DNA
content, the sub-G1 population, which is generally regarded as
representing cells that have undergone apoptotic cell death. As
shown for examples in Fig. 1C and summarized in Fig. 1D, Cd
induced a concentration-dependent increase in the sub-G1 population up to concentrations of 200 M in RTgill-W1 cells and up
to 500 M in the RTH-149 cells (supplementary Fig. S1). In contrast, Cu caused no signicant increase of the sub-G1 population in
either cell type irrespective of the concentration applied (Figs. 1C
and S1).

ciated with typical cell swelling (supplementary movie S1). In

contrast, with 200 M Cu only mild morphological cellular changes
were evident and also nuclear appearance seemed unaltered in
RTgill-W1 cells (Fig. 2). A mixed apoptotic/necrotic death phenotype was also observed in RTH-149 hepatoma cells exposed to
Cd at 50 M and 200 M (Fig. S2 and movie S2). For comparison, movies of primarily necrotic death occurring during exposure
to 500 M Cd and of the onset of apoptotic death after addition of 0.2 M of the pan-kinase-inhibitor staurosporine (STS) are
shown as supplementary movies S6 and S7 (also compare stills in
Fig. S2, right panels).

3.2. Cellular and nuclear morphology

3.3. Mitochondrial membrane potential (MMP)

Morphological alterations of gill cells determined at the end

of the 120 h incubation period with 50 M Cd indicated partly
apoptotic features, as evidenced by retracting but still attached
cell bodies and heavily condensed or fragmented nuclei, and this
was even more pronounced at 200 M Cd (Fig. 2). However, the
number of apparently apoptotic nuclei could not account for the
decrease of viability assessed by the MTT assay and when morphological changes were followed over time, using time-lapse imaging,
it became clear that many cells underwent necrotic death asso-

While changes of cell viability or DNA contents took relatively

long to become apparent, one may expect that pathways signaling or mediating cell death should be detectable at earlier time
points. As such, alterations of MMP may be an early event and
indeed we found that after only 24 h a concentration-dependent
increase in the population of gill cells with diminished MMP was
evident (Fig. 3A). At 48 h of incubation with 50 M Cd individual
cells with condensed perinuclear mitochondria could be observed
(Fig. 3B) and after 72 h 23% (50 M Cd) to 55% (500 M Cd) of the

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

77

Fig. 2. Cellular and nuclear morphology of RTgill-W1 cells exposed to Cd or Cu for 120 h. Typical examples of the light microscopic appearance of gill cells exposed as
indicated, and of their DAPI-stained nuclei as observed by uorescence microscopy.

cell population had a low MMP, compared to 13% in the controls

(Fig. 3C and D). In Cu-treated cells no comparable changes were evident at these time points as determined by measuring the retention
of TMRM (not shown).
3.4. Induction of DNA damage response markers
Another early event induced by toxic levels of metals is the
appearance of DNA damage. In the present context this was of
particular interest, as the intrinsic leg of programmed necrotic cell
death was shown to be triggered upon activation of the DNA repair
enzyme poly-(ADP-ribose) polymerase-1 (PARP-1), which by not
yet clearly dened mechanisms may activate RIP-1 and in consequence necroptosis (Festjens et al., 2006). We thus tested whether
molecules regarded as typical markers of the DNA damage response
would be activated upon exposure of the sh cells to Cd or Cu. As
a control treatment known to induce DNA double-strand breaks,

serving to assure DNA damage can be detected with the mammalian protein-directed antibodies applied, we exposed the sh
cells to -irradiation. As depicted in Fig. 3, both -irradiation as well
as incubation with Cd or Cu activated the DNA damage response
protein Ataxia telangiectasia mutated (ATM) to its phosphorylated
form, phospho-ATM, as well as one of its downstream targets, histone H2A, to the active, phosphorylated form, -H2AX (Figs. 4A
and S2). This was not only detected in individual cells, but also
evident as a right-shift of -H2AX staining intensity at the population level determined by FACS analysis (Fig. 4B). These changes
were already evident at 8 h of incubation (Fig. 4A) and persisted
through at least 24 h of metal exposure (Figs. 4B and S2). Another
molecule downstream of ATM, Checkpoint kinase 2 (Chk-2), was
also found to be activated by both irradiation and metal exposure (Fig. 4C). Together, these observations support that a DNA
damage response is readily elicited upon incubation with Cd or
Cu.

78

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Fig. 3. Mitochondrial membrane potential (MMP) of RTgill-W1 cells exposed to Cd. Gill cells were exposed to Cd and changes of their MMP were estimated by use of the
potential-sensitive uorescence dye JC-1. Panel (A) depicts examples of FACS plots of cells exposed to Cd at the indicated concentrations for 24 h, with the left images showing
dot plots of highly energized mitochondria (J-aggregates; FL-2) versus mitochondrial with low potential (J monomers; FL-1) and the right images showing the frequency
distribution of red uorescence. In (B) examples of JC-1 loaded individual cells are shown after exposure to control conditions or to Cd for 48 h, with red J-aggregates in the
upper panels and green monomers in the lower panels. Panel (C) shows examples for the decrease of MMP, estimated from the decrease of J-aggregate uorescence, in cells
incubated with Cd for 72 h, and in (D) means SE of 3 independent experiments are summarized.

3.5. Caspase-3 activity

One of the hallmarks of classical apoptosis, often triggered in
response to failed DNA damage response, is activation of the executioner caspase-3. Indeed, in RTgill-W1 cells 50 M Cd elicited an
activation of the protease from day 3 onwards, whereas 200 M Cd
caused only a slight transient elevation at day 1, but no enhanced
caspase-3 activity during the subsequent days (Fig. 5A). A complete
lack of caspase activation was noted with 200 M Cu, while, for
comparison, the typical apoptosis inducing agent STS stimulated
a pronounced increase within as little as 12 h. A slightly different
picture was seen in the hepatoma cells, in which both 50 M and
200 M Cd induced a transient elevation of caspase-3 activity during the rst 2 days followed by a return to and even below basal
levels (Fig. 5B). Furthermore, in these cells Cu caused a pronounced,
albeit transient increase of caspase-3 activity at day 3, whereas
STS, again, evoked a rapid elevation of similar magnitude within
12 h.

3.6. The impact of necrostatin-1 (Nec-1) and caspase-inhibition

on cell death
Based on the ndings described so far, it appeared that both
apoptosis and necrosis-like death occurred in the sh cells upon
metal exposure. In order to estimate the relative contribution of
either cell death mode and to elucidate if necroptosis is involved
in this scenario, cells were exposed to the metals in the absence
and presence of the pan-caspase inhibitor zVAD-fmk or the RIP-1
inhibitor Nec-1. Further, to reduce unwanted effects such as secondary necrosis, which particularly in vitro may follow apoptosis
in the absence of phagocyting cells (Vandenabeele et al., 2006),
cell viability was already assessed after 72 h of incubation, a time
when caspase-3 has already been activated or activation was maximal when present (see Fig. 5). As shown in Fig. 6A for the gill
cell line and assessed with the MTT assay, both Nec-1 and zVADfmk conferred signicant protection against 50 M Cd, although
the effect was clearly less pronounced with the caspase inhibitor.

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

79

Fig. 4. Induction of DNA damage response proteins in RTH-149 cells. Trout hepatoma cells were exposed to 30 grey -irradiation, 50 M Cd, or 200 M Cu and then xed
and immunostained for -H2AX and phospho-ATM (A) or phospho-Chk-2 (C) and counterstained with DAPI as nuclear marker. Scale bars indicate 20 m. In panel (B) results
of a FACS analysis of cells exposed to Cd are depicted, showing the right-shift of uorescence intensity reecting the increase of -H2AX levels in the cell population.

The same was observed when PI-positive cells were examined, in

which the increase to 34% PI-positive cells observed with 50 M
Cd was reduced to a mean of 12% and 17% with Nec-1 and zVADfmk, respectively (Fig. 6B). Partial protection was still seen with
Nec-1 in the presence of 200 M Cd (MTT assay), whereas zVADfmk no longer prevented cell death at this concentration. Better
preservation of cellular and nuclear morphology was even visible
after 120 h of incubation with 50 M Cd, but clearly no more with
200 M (Fig. S4). There was no synergistic effect of both inhibitors
detectable against Cd toxicity (Fig. S5) and when Nec-1 was applied
at higher concentrations it became toxic by itself (not shown). In
comparison, in cells treated with Cu no protection at all, but rather
an aggravation of cell death was observed when inhibitors were
applied separately (Fig. 6A) or concurrently (not shown).
Basically similar results were obtained with another viability
assay which is in fact based on cellular ATP contents (Fig. 6C). A
notable difference was that Nec-1 was not protective against the
loss of ATP at 200 M and that Cu had apparently no impact on ATP
at all when given alone. In a further experimental series we determined MMP, as reected by the retention capability of TRMR, and
obtained yet again results with a similar tendency, although most
changes were largely dampened and thus not signicant (Fig. S5B).

In RTH-149 cells, again examined with the MTT assay, we

made qualitatively similar observations as in the gill cells in
that Nec-1 conferred apparent partial protection against Cd,
but did not protect or even aggravated Cu-toxicity (Fig. S6A).
Unfortunately, Nec-1 appeared to be toxic by itself to these
cells at a concentration when it was effective, reducing viability by 20% within 3 days. Its protective effect was thus
only signicant at 200 M Cd, but not at 50 M. The caspase
inhibitor zVAD-fmk did not signicantly protect against any treatment, although similar tendencies were seen as with the gill
cells.
3.7. The impact of Nec-1 and caspase-inhibition on caspase
activity and ROS formation
As shown in Fig. 7A, caspase-inhibition with zVAD-fmk worked
perfectly well even over 72 h, since there was no increase of caspase
activity seen in its presence irrespective of the metal and its concentration present. In contrast, Nec-1 had no signicant impact on
caspase activity in any treatment, although it caused a slight reduction of the activation seen in the presence of 50 M Cd. In line with
this, we also observed a protective effect in terms of the number

80

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Fig. 5. Caspase-3 activity of trout cells exposed to Cd or Cu. RTgill-W1 (A) and
RTH-149 (B) cells were exposed to Cd or Cu at the concentrations indicated and
caspase activity was estimated from the increase of uorescence upon cleavage
of zDEVD-R110 after 05 days of exposure. For comparison, caspase activity was
also determined in cells incubated with the pan-kinase-inhibitor staurosporine (STS,
0.5 M) for 12 h. Data are means SE of 3 cultures. *p < 0.05 versus control at time
zero.

of sub-G1 cells, the small increase of which, although not statistically signicant, was totally blocked by both Nec-1 or by zVAD-fmk
(Fig. S5C).
Besides inhibiting caspases, zVAD-fmk was shown to interact
with components of the mitochondrial permeability transition pore
(MPTP) and to thereby promote necrotic cell death (Temkin et al.,
2006). In order to exclude that this masked the effect of caspaseinhibition, we also tested QVD, a caspase inhibitor with a different
chemical backbone (Caserta et al., 2003). However, viability of gill
cells treated with 50 M Cd in the absence and presence of 20 M
QVD amounted to 59 5% and 41 6% (n = 4), respectively. The
same lack of protection was also indicated in time-lapse movies
made in the presence of the inhibitor (not shown), suggesting that
zVAD-fmk is a more potent inhibitor of teleost caspases.
The induction of radical production has been described to
accompany exposure to Cd or Cu (Manzl et al., 2004; Risso-de
Faverney et al., 2004) and also repeatedly upon induction of necroptosis (Goossens et al., 1999). Unexpectedly, however, we could not
detect an increase of ROS formation with any of the treatments
applied, neither after 6 h of exposure (Fig. 7B), nor after 24 h (not
shown).

Fig. 6. The impact of inhibition of necroptosis or caspase activity on cell viability

of RTgill-W1 cells exposed to Cd or Cu. Gill cells were pre-incubated with 10 M
necrostatin-1 (Nec-1) or zVAD-fmk (zVAD) for 1 h and then incubated with Cd or
Cu, in the continued presence of inhibitors, for 72 h. At the end of the period cell
viability was estimated by use of the MTT assay (A), by determining PI-uptake (B),
or with a viability assay based on cellular ATP contents (C). Data are means SE of

37 cultures. *p < 0.05 versus control, p < 0.05 versus metal only.

3.8. Effects of other inhibitors of necroptotic and apoptotic

pathways
In addition to RIP, which can not only be directly blocked by use
of Nec-1, but also indirectly by interfering with its molecular chap-

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Fig. 7. The impact of inhibition of necroptosis or caspase activity on caspase activity

and ROS formation of RTgill-W1 cells exposed to Cd or Cu. Gill cells were preincubated with 10 M Nec-1 or zVAD for 1 h and then further incubated with Cd or
Cu, in the continued presence of inhibitors, for 72 h (A) or 6 h (B). At the end of the
exposure time caspase activity (A) or the formation of ROS (B) were estimated with
uorescence-based assays. Cells treated with 1 mM H2 O2 for 1 h were included as a
positive control. Data are means SE of 3 and 8 (3 for H2 O2 ) cultures, respectively.
*p < 0.05 versus control.

erone hsp-90, preventing its proteasomal degradation (Lewis et al.,

2000), other suggested molecular constituents of the necroptotic
pathway are the DNA repair enzyme PARP-1 and the mitogenactivated protein kinase JNK (Deng et al., 2003). Furthermore, it
seems that the MPTP or at least a component of it, cyclophilin D,
may be involved in necroptosis (Baines et al., 2005; Nakagawa et
al., 2005). We thus tested the impact of specic inhibitors of these
molecules to assess if any of these ndings can be veried for the
sh cells exposed to Cd.
In fact, it appeared that inhibition of hsp-90 function had
qualitatively similar effects as Nec-1, but since it exerted considerable toxicity against the RTgill-W1 cells by itself, this protective
effect was not signicant (Fig. 8A). In comparison, in the RTH-149
cells, for which it was apparently less toxic, it conferred complete protection against 50 M Cd, although not against 200 M
(Fig. 8B). The competitive PARP inhibitor 3-aminobenzamide, a
nicotinamide analogue, had no effect on cell viability of gill
cells or hepatoma cells, and the same was true for cyclosporine
A, an inhibitor of the MPTP component cyclophilin D (Fig. 8).
Finally, we also tested inhibitors of the MAP kinase JNK and

81

Fig. 8. The impact of inhibitors of hypothetical necroptosis pathway proteins on viability of RTgill-W1 (A) or RTH-149 cells (B) exposed to Cd. Cells were pre-incubated
for 1 h with 1 M 17-DMAG (hsp-90 inhibitor), 0.5 mM 3-aminobenzamide (PARP
inhibitor), or 10 M cyclosporine A (PTP inhibitor). Then cells were exposed to Cd,
in the continued presence of inhibitors, for 72 h and cell viability was assessed from
MTT conversion. Data are means SE of 11-12 (A) and 4-8 (B) cultures. *p < 0.05
versus control.

of MAP kinases p38 and ERK. Although only JNK and not the
latter ones have been implied in necroptosis, they play multiple roles in apoptosis (Wada and Penninger, 2004). However,
all these treatments, and in particular inhibition of the generally anti-apoptotic ERK were toxic even in the absence of Cd and
thus none of these treatments was benecial for cell survival
(Fig. S6B).
3.9. Ultrastructural changes induced by Cd and Cu
In a nal series of experiments we examined morphological
changes of RTH-149 cells using a rapid cryo-xation that avoids
xation artifacts. As depicted in Fig. 9A, controls had homogeneous
chromatin in a round-shaped nucleus, elongated mitochondria
with clearly discernible cristae, a modest number of lysosomes
and a well-dened Golgi complex with multiple attened cisternal
membranes. In cells exposed to 200 M Cu for 48 h, there were no
major apparent changes visible, although the number of electronlucent vesicles seemed to have increased in some cells (Fig. 9B). In

82

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Fig. 9. Ultrastructural changes of RTH-149 cells exposed to Cd or Cu. Hepatoma cells were incubated under control conditions (A) or with 200 M Cu (B) or 50 M Cd (C)
for 48 h, xed by high-pressure freezing and analyzed by transmission electron microscopy. In (D) controls and Cd-treated cells were immunostained for the Golgi marker
GM-130 and counterstained with the nuclear marker DAPI. The lowermost image is an enlargement of the cell marked with a white rectangle in the middle panel.

contrast, while the nuclear morphology was still intact upon 48 h

exposure to Cd, the Golgi apparatus appeared profoundly dilated
and seemed to be en route to total disintegration. Given that the
latter nding was so prominent, we also tested changes of the
Golgi structure by immunouorescence and found the EM ndings
conrmed. Thus, whereas the FITC-labeled antibody for the Golgi
marker GM-130 was nicely localized in the perinuclear region in
the controls (Fig. 9D) and in Cu-treated cells (Fig. S7), it stained a
compartment that was largely fragmented and partly more distant
from the nucleus in cells exposed to 50 M and 200 M Cd (Figs. 9D
and S7).

4. Discussion
4.1. Cd induces multiple modes of cell death in trout cell lines
Rainbow trout cell lines exposed to moderate levels of Cd underwent cell death that at rst sight seemed to primarily show signs
of classical apoptosis. That is, upon addition of Cd cells very slowly
(after >48 h) lost cellcell contacts and retracted from the substratum and at the end of 96120 h of incubation many cells displayed
heavily condensed and/or fragmented nuclei as well as roundedup cell bodies. Furthermore, we saw an increase in the sub-G1

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

population typically reecting apoptotic cells and in most conditions at least a slight transient elevation of caspase-3 activity was
observed. At the same time, however, the decrease of cell viability detected by the MTT assay (specically 50% viability at 50 M
Cd) was quantitatively not at all accounted for by the number of
cells with an apoptotic nucleus (which was not quantied) or by
the percentage of sub-G1 cells (an increase of 5% at 50 M Cd).
In contrast, the increase of PI-positive cells largely agreed with
diminished MTT conversion capacity. In addition, if caspase activity
was elevated this was generally moderate, and when morphological changes were followed over time it became apparent that
numerous cells displayed cell swelling and not typical apoptotic
blebbing. Together, these ndings indicate that most cells died
not by apoptosis but rather by some form of necrosis. In support
for this notion, we observed a relatively early decrease of MMP
in many cells and a pronounced decline of cellular ATP contents,
which is usually considered to reect necrotic cell death (Eguchi et
al., 1997). Most importantly, however, the decrease of cell viability,
estimated with MTT and PI-uptake, and the loss of ATP were to a
signicant extent inhibited by the RIP-1 inhibitor Nec-1 and, generally only to a lesser degree, reduced by the pan-caspase inhibitor
zVAD-fmk. By the denition used in the present study, Cd-exposed
trout cells thus died by a mix of necroptosis and apoptosis, i.e.
by Nec-1 sensitive and by zVAD-fmk sensitive cell death, respectively. The occurrence of secondary necrosis seems unlikely in this
context, as this was recently shown to be insensitive to Nec-1
(Berghe et al., in press). The mixed necroptotic/apoptotic nature
of cell death was generally more obvious at 50 M Cd, whereas at
200 M zVAD-fmk no longer signicantly affected the parameter in
question. In line, caspase activation was absent (gill cells) or moderate (hepatoma cells) at this concentration and the only indication
of apoptotic death was the occurrence of apoptotic nuclear morphology and the increase in sub-G1 cells. Noteworthy, at 200 M
Cd the latter would in fact agree with the decrease of viability as
estimated from the MTT assay, but if sub-G1 cells truly reected
apoptotic death, then this must have been a caspase-independent
form of apoptosis. Furthermore, in this case PI would then have
labeled cells following secondary necrosis. In support for this, Cdtriggered caspase-independent apoptosis has been described for
many cell models, e.g. rat kidney proximal tubule cells (Lee et al.,
2006), rat hepatocytes (Pham et al., 2006), a human lymphoblastoid
cell line (Coutant et al., 2006) and mouse mesangial cells (Liu and
Templeton, 2008), in each case associate with and possibly mediated by the mitochondrial release of apoptosis inducing factor (AIF),
an oxidoreductase that is involved in chromatin condensation and
DNA fragmentation (Cande et al., 2004). But even if this also happened in the trout cells, there was still an additional component
of Nec-1 sensitive cell death observed at 200 M Cd, indicating
that while necroptosis persisted also at elevated Cd concentration,
the caspase-dependent component of apoptosis vanished. Furthermore, at both concentrations cell death was seen that could neither
be blocked by Nec-1 nor by zVAD-fmk (or a combination of both),
suggesting that caspase-independent apoptosis may also have contributed at the lower Cd concentration. Taken together, our data
are in line with previous reports of multiple modes of cell death
induced by Cd and extend these observations by adding necroptosis
as one pathway besides others.
4.2. A DNA damage response is triggered by Cd and may initiate
cell death
The mechanisms by which Cd may induce cell death are manifold and include enhanced formation of radicals (Liu et al., 2009;
Pathak and Khandelwal, 2006; Risso-de Faverney et al., 2001), damage of mitochondria with ensuing opening of the MPTP (Belyaeva
et al., 2006) or with the release of apoptogenic factors independent

83

of this pore (Lee et al., 2005), disturbance of ion homeostasis, in

particular of Ca2+ (Yang et al., 2007), activation of proteases (Hsu et
al., 2009; Lee et al., 2007), and the induction of DNA damage (Bertin
and Averbeck, 2006; Viau et al., 2008). Many of these mechanisms
are closely intertwined and it is often not generally clear which
process is upstream or downstream of others. In the case of the
trout cell lines, a primary role for radicals appears unlikely, as no
enhanced ROS formation was detected within the rst 24 h of exposure. It should be mentioned, however, that ROS formation induced
by Cd may also be a transient phenomenon (Thvenod, 2009) and
thus we cannot completely rule out that such a transient elevation of radicals was overlooked. In contrast, we clearly detected
the activation of proteins involved in the DNA damage response,
i.e. of H2AX, phospo-ATM and phospho-Chk-2, within a timeframe when no other effects of the metal were detectable. In line,
it has been reported that 30 M Cd may induce DNA double-strand
breaks, leading to the appearance of H2AX foci, within as little as
1 h in a human endothelial cell line (Viau et al., 2008). A similarly
rapid induction of DNA damage, as judged from the appearance
of Comet tails, has recently been described for rat kidney proximal tubule cells exposed to 50100 M Cd, and this then led to
Chk-1/2 mediated arrest in G2/M phase (Bork et al., 2010). Such
an arrest was apparently not induced in the trout cells, since timelapse movies indicated that the cells continued to proliferate for
about 48 h before cell damage became evident.
As outlined in the introduction, extensive DNA damage may
in fact trigger what could be referred to as intrinsic necroptosis
(Festjens et al., 2006; Xu et al., 2006; Zong et al., 2004). Specically, it has been suggested that overactivation of PARP could lead
to depletion of cytosolic NAD levels and in consequence of cellular
ATP (Zong et al., 2004), setting in motion a still ill-dened necroptotic cascade. The decrease of ATP observed in the sh cells would
agree with such a scenario. However, besides directly damaging
DNA, Cd has also been shown to cause genotoxic stress through
inhibition of DNA damage repair enzymes (Bertin and Averbeck,
2006; Whiteside et al., 2010), including PARP activity (Hartwig et
al., 2002). Thus, enhanced PARP activity may have little effect on
ATP levels in Cd-exposed sh cells, which would agree with the
observation that inhibition of the enzyme had no effect on cell viability. Upon prolonged exposure to Cd, the suppression of PARP and
other repair enzymes will nonetheless become a main issue as DNA
damage will accumulate in the cells to an extent that is no longer
compatible with proper cell function and ultimately survival.
4.3. Further peculiarities of Cd-induced cell death of trout cells
Irrespective of the role of PARP, it was clear that inhibition of RIP1, either directly with Nec-1 or indirectly by blocking its molecular
chaperone hsp-90, conferred partial protection against Cd-induced
cell death. This is in contrast to the report by Hsu et al. (2009), where
Nec-1, but not geldanamycin, an analogue of the hsp-90 inhibitor
used here, protected Chinese hamster ovary cells against Cd, a discrepancy which remains unexplained at present. In contrast to the
unequivocal role of RIP-1, other molecular constituents of programmed necrosis previously identied could not be extended to
teleost cells here, as, e.g. the prolonged inhibition of JNK (as well as
of ERK and p38) was toxic by itself, and inhibition of the MPTP component cyclophilin D with CsA was without effect on cell viability.
The latter appeared unexpected considering that a decrease of MMP
during necrosis-like cell death is often the result of MPTP opening
(Kim et al., 2003). On the other hand, the role of the mitochondria in necroptosis is still largely unclear (Christofferson and Yuan,
2010) and may even involve classical apoptotic molecules such as
Bmf (Hitomi et al., 2008), suggesting possible links between apoptosis and necroptosis. In this context it seems noteworthy that in
the Cd-treated sh cells, differently from other reports (Han et al.,

84

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

2009), inhibition of either necroptosis or of caspase activity did not

result in the elevation of the respective uninhibited pathway. Thus,
neither did the presence of Nec-1 enhance apoptotic features (i.e.
caspase activity, sub-G1 cells, fragmented nuclei), nor did zVADfmk increase the number of PI-positive cells or the drop in ATP
levels. Curiously, it rather seemed that Nec-1, at least to a small but
not signicant extent, reduced the above named apoptotic features
when present. Thus, despite of the lack of an additive protective
effect of Nec-1 and zVAD-fmk under the conditions tested, there
appeared to be some crosstalk, the nature and importance of which
has yet to be addressed in future studies.
A nal peculiarity of Cd-induced cell death in trout cells
detected here is the early appearance of Golgi disintegration,
occurring before the detection of any other sub-cellular morphological alterations. Previously, an involvement of the Golgi
complex was reported for receptor-mediated apoptosis or necrosis, where it played a role in the activation of ceramide-generating
acid-sphingomyelinase and the lysosomal protease cathepsin D
(Schneider-Brachert et al., 2004), and in cytosolic phospholipase
A2-mediated necrosis (Festjens et al., 2006). Given that the Golgi
complex may act as a sensor and transducer of stress signals in various settings (Hicks and Machamer, 2005), a more detailed look into
its potential role in necroptosis is clearly warranted.
4.4. The apparent lack of Cu-toxicity
In previous studies on primary trout hepatocytes we had
observed that the acute toxicity of Cu exceeded that of Cd (Manzl
et al., 2003) and that Cu caused opening of the MPTP and induced
cell death with a similar mixed phenotype as observed here for Cd
(Krumschnabel et al., 2005; Nawaz et al., 2006). Surprisingly, this
was not at all recapitulated in the trout cell lines used here and
compared to Cd much higher concentrations of Cu were required
to induce cell death. Furthermore, cell death was characterized by
the absence of apoptotic markers (except from a transient elevation of caspase activity in RTH-149 cells) and by a relatively minor
increase in PI-positive cells and, most conspicuously, incubation
with Nec-1 or zVAD-fmk aggravated cell death instead of ameliorating it. Together, this suggests that necroptosis is not induced in the
Cu-exposed trout cell lines and that, differently from primary hepatocytes (Nawaz et al., 2006), also the inhibition of caspase activity
does not confer any protection. The exact mode of Cu-induced cell
death thus remains open here and it appears that much higher Cu
levels will be required to induce faster cell death which is more
likely to involve programmed or secondary necrosis (Festjens et
al., 2006).
4.5. Cell type specic responses
A previous study has shown higher resistance at low concentrations and greater sensitivity at high concentrations towards an
environmentally relevant toxicant of RTL-W1 liver cells, another
trout-derived liver cell line, as compared to RTgill-W1 cells (Shaoa
et al., 2008). Here, we observed that the gill cell line was generally less sensitive to Cd and Cu than RTH-149 cells, dying at a
slower rate and/or at higher concentration of the metals. The use
of cell lines does quite obviously not perfectly mirror the situation in primary cells from the organs they were derived from and
these differences underscore that extrapolation of these in vitro
ndings to an ecologically relevant situation is most likely not feasible. However, as such cell lines guarantee unlimited provision of
cells with minimal biological variability, their use for unraveling
principle mechanisms sure makes them an attractive and useful
alternative to the much more limited and labor-intensive use of
primary cell cultures.

Acknowledgements
We thank Karin Gutleben for excellent technical assistance. GK
and AV were supported by a grant from the Austrian Science Fund
(FWF; Y212-B13 START). HLE was supported by grants from the
Austrian Science Funds (FWF-P19486-B12) and the Tiroler Wissenschaftsfonds (P-UNI-0404/100) to M.W. Hess.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.aquatox.2010.04.005.
References
Baines, C.P., Kaiser, R.A., Purcell, N.H., Blair, N.S., Osinska, H., Hambleton, M.A., Brunskill, E.W., Sayen, M.R., Gottlieb, R.A., Dorn, G.W., et al., 2005. Loss of cyclophilin
D reveals a critical role for mitochondrial permeability transition in cell death.
Nature 434, 658662.
Belyaeva, E.A., Dymkowska, D., Wieckowski, M.R., Wojtczak, L., 2006. Reactive oxygen species produced by the mitochondrial respiratory chain are involved in
Cd2+ -induced injury of rat ascites hepatoma AS-30D cells. Biochim. Biophys.
Acta 1757, 15681574.
Berghe, T.V., Vanlangenakker, N., Parthoens, E., Deckers, W., Devos, M., Festjens,
N., Guerin, C.J., Brunk, U.T., Declercq, W., Vandenabeele, P., in press. Necroptosis, necrosis and secondary necrosis converge on similar cellular disintegration
features. Cell Death Differ.
Bertin, G., Averbeck, D., 2006. Cadmium: cellular effects, modications of
biomolecules, modulation of DNA repair and genotoxic consequences (a review).
Biochimie 88, 15491559.
Bork, U., Lee, W.K., Kuchler, A., Dittmar, T., Thevenod, F., 2010. Cadmium-induced
DNA damage triggers G(2)/M arrest via chk1/2 and cdc2 in p53-decient kidney
proximal tubule cells. Am. J. Physiol. Renal Physiol. 298, F255F265.
Cande, C., Vahsen, N., Garrido, C., Kroemer, G., 2004. Apoptosis-inducing factor (AIF):
caspase-independent after all. Cell Death Differ. 11, 591595.
Caserta, T.M., Smith, A.N., Gultice, A.D., Reedy, M.A., Brown, T.L., 2003. Q-VD-OPh, a
broad spectrum caspase inhibitor with potent antiapoptotic properties. Apoptosis 8, 345352.
Christofferson, D.E., Yuan, J., 2010. Necroptosis as an alternative form of programmed
cell death. Curr. Opin. Cell Biol. 22, 263268.
Coutant, A., Lebeau, J., Bidon-Wagner, N., Levalois, C., Lectard, B., Chevillard, S.,
2006. Cadmium-induced apoptosis in lymphoblastoid cell line: involvement of
caspase-dependent and -independent pathways. Biochimie 88, 18151822.
Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N., Cuny, G.D., Mitchison, T.J., Moskowitz, M.A., Yuan, J., 2005. Chemical inhibitor of nonapoptotic cell
death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 1,
112119.
Deng, Y., Ren, X., Yang, L., Lin, Y., Wu, X., 2003. A JNK-dependent pathway is required
for TNFalpha-induced apoptosis. Cell 115, 6170.
Eguchi, Y., Shimizu, S., Tsujimoto, Y., 1997. Intracellular ATP levels determine cell
death fate by apoptosis or necrosis. Cancer Res. 57, 18351840.
Festjens, N., Vanden Berghe, T., Vandenabeele, P., 2006. Necrosis, a well-orchestrated
form of cell demise: signalling cascades, important mediators and concomitant
immune response. Biochim. Biophys. Acta 1757, 13711387.
Goossens, V., De Vos, K., Vercammen, D., Steemans, M., Vancompernolle, K., Fiers, W.,
Vandenabeele, P., Grooten, J., 1999. Redox regulation of TNF signaling. Biofactors
10, 145156.
Habeebu, S.S., Liu, J., Klaassen, C.D., 1998. Cadmium-induced apoptosis in mouse
liver. Toxicol. Appl. Pharmacol. 149, 203209.
Han, W., Xie, J., Li, L., Liu, Z., Hu, X., 2009. Necrostatin-1 reverts shikonin-induced
necroptosis to apoptosis. Apoptosis 14, 674686.
Hartwig, A., Asmuss, M., Blessing, H., Hoffmann, S., Jahnke, G., Khandelwal, S.,
Pelzer, A., Burkle, A., 2002. Interference by toxic metal ions with zinc-dependent
proteins involved in maintaining genomic stability. Food Chem. Toxicol. 40,
11791184.
Hess, M.W., Muller, M., Debbage, P.L., Vetterlein, M., Pavelka, M., 2000. Cryopreparation provides new insight into the effects of brefeldin A on the structure of the
HepG2 Golgi apparatus. J. Struct. Biol. 130, 6372.
Hicks, S.W., Machamer, C.E., 2005. Golgi structure in stress sensing and apoptosis.
Biochim. Biophys. Acta 1744, 406414.
Hitomi, J., Christofferson, D.E., Ng, A., Yao, J., Degterev, A., Xavier, R.J., Yuan, J., 2008.
Identication of a molecular signaling network that regulates a cellular necrotic
cell death pathway. Cell 135, 13111323.
Hsu, T.S., Yang, P.M., Tsai, J.S., Lin, L.Y., 2009. Attenuation of cadmium-induced
necrotic cell death by necrostatin-1: potential necrostatin-1 acting sites. Toxicol.
Appl. Pharmacol. 235, 153162.
Joseph, P., 2009. Mechanisms of cadmium carcinogenesis. Toxicol. Appl. Pharmacol.
238, 272279.
Kim, J.S., He, L., Lemasters, J.J., 2003. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem. Biophys. Res. Commun. 304,
463470.

G. Krumschnabel et al. / Aquatic Toxicology 99 (2010) 7385

Krumschnabel, G., Manzl, C., Berger, C., Hofer, B., 2005. Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes.
Toxicol. Appl. Pharmacol. 209, 6273.
Krumschnabel, G., Podrabsky, J.E., 2009. Fish as model systems for the study of
vertebrate apoptosis. Apoptosis 14, 121.
Lee, W.K., Abouhamed, M., Thevenod, F., 2006. Caspase-dependent and independent pathways for cadmium-induced apoptosis in cultured kidney
proximal tubule cells. Am. J. Physiol. Renal Physiol. 291, F823F832.
Lee, W.K., Bork, U., Gholamrezaei, F., Thevenod, F., 2005. Cd(2+)-induced cytochrome
c release in apoptotic proximal tubule cells: role of mitochondrial permeability
transition pore and Ca(2+) uniporter. Am. J. Physiol. Renal Physiol. 288, F2739.
Lee, W.K., Torchalski, B., Thevenod, F., 2007. Cadmium-induced ceramide formation
triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells.
Am. J. Physiol. Cell Physiol. 293, C839C847.
Lewis, J., Devin, A., Miller, A., Lin, Y., Rodriguez, Y., Neckers, L., Liu, Z.G., 2000.
Disruption of hsp90 function results in degradation of the death domain
kinase, receptor-interacting protein (RIP), and blockage of tumor necrosis factorinduced nuclear factor-kappaB activation. J. Biol. Chem. 275, 1051910526.
Liu, J., Qu, W., Kadiiska, M.B., 2009. Role of oxidative stress in cadmium toxicity and
carcinogenesis. Toxicol. Appl. Pharmacol. 238, 209214.
Liu, Y., Templeton, D.M., 2008. Initiation of caspase-independent death in mouse
mesangial cells by Cd2+ : involvement of p38 kinase and CaMK-II. J. Cell. Physiol.
217, 307318.
Lopez, E., Figueroa, S., Oset-Gasque, M.J., Gonzalez, M.P., 2003. Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture. Br.
J. Pharmacol. 138, 901911.
Lyons-Alcantara, M., Mooney, R., Lyng, F., Cottell, D., Mothersill, C., 1998. The effects
of cadmium exposure on the cytology and function of primary cultures from
rainbow trout. Cell Biochem. Funct. 16, 113.
Manzl, C., Ebner, H., Kock, G., Dallinger, R., Krumschnabel, G., 2003. Copper, but not
cadmium, is acutely toxic for trout hepatocytes: short-term effects on energetics
and ion homeostasis. Toxicol. Appl. Pharmacol. 191, 235244.
Manzl, C., Enrich, J., Ebner, H., Dallinger, R., Krumschnabel, G., 2004. Copper-induced
formation of reactive oxygen species causes cell death and disruption of calcium
homeostasis in trout hepatocytes. Toxicology 196, 5764.
Nakagawa, T., Shimizu, S., Watanabe, T., Yamaguchi, O., Otsu, K., Yamagata, H., Inohara, H., Kubo, T., Tsujimoto, Y., 2005. Cyclophilin D-dependent mitochondrial
permeability transition regulates some necrotic but not apoptotic cell death.
Nature 434, 652658.
Nawaz, M., Manzl, C., Lacher, V., Krumschnabel, G., 2006. Copper-induced stimulation of extracellular signal-regulated kinase in trout hepatocytes: the role of
reactive oxygen species, Ca2+ , and cell energetics and the impact of extracellular signal-regulated kinase signaling on apoptosis and necrosis. Toxicol. Sci. 92,
464475.
Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., Riccardi, C., 1991. A rapid
and simple method for measuring thymocyte apoptosis by propidium iodide
staining and ow cytometry. J. Immunol. Methods 139, 271279.
Pathak, N., Khandelwal, S., 2006. Oxidative stress and apoptotic changes in murine
splenocytes exposed to cadmium. Toxicology 220, 2636.
Pham, T.N., Marion, M., Denizeau, F., Jumarie, C., 2006. Cadmium-induced apoptosis
in rat hepatocytes does not necessarily involve caspase-dependent pathways.
Toxicol. In Vitro 20, 13311342.

85

Pulido, M.D., Parrish, A.R., 2003. Metal-induced apoptosis: mechanisms. Mutat. Res.
533, 227241.
Risso-de Faverney, C., Devaux, A., Lafaurie, M., Girard, J.P., Bailly, B., Rahmani, R.,
2001. Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species. Aquat. Toxicol. 53,
6576.
Risso-de Faverney, C., Orsini, N., de Sousa, G., Rahmani, R., 2004. Cadmium-induced
apoptosis through the mitochondrial pathway in rainbow trout hepatocytes:
involvement of oxidative stress. Aquat. Toxicol. 69, 247258.
Sancho, P., Fernandez, C., Yuste, V.J., Amran, D., Ramos, A.M., de Blas, E., Susin, S.A.,
Aller, P., 2006. Regulation of apoptosis/necrosis execution in cadmium-treated
human promonocytic cells under different forms of oxidative stress. Apoptosis
11, 673686.
Shaoa, J., Eckerta, M.L., Leeb, L.E.J., Gallagher, E.P., 2008. Comparative oxygen radical
formation and toxicity of BDE 47 in rainbow trout cell lines. Mar. Environ. Res.
66, 78.
Schneider-Brachert, W., Tchikov, V., Neumeyer, J., Jakob, M., Winoto-Morbach, S.,
Held-Feindt, J., Heinrich, M., Merkel, O., Ehrenschwender, M., Adam, D., et
al., 2004. Compartmentalization of TNF receptor 1 signaling: internalized TNF
receptosomes as death signaling vesicles. Immunity 21, 415428.
Shih, C.M., Wu, J.S., Ko, W.C., Wang, L.F., Wei, Y.H., Liang, H.F., Chen, Y.C., Chen,
C.T., 2003. Mitochondria-mediated caspase-independent apoptosis induced by
cadmium in normal human lung cells. J. Cell. Biochem. 89, 335347.
Temkin, V., Huang, Q., Liu, H., Osada, H., Pope, R.M., 2006. Inhibition of ADP/ATP
exchange in receptor-interacting protein-mediated necrosis. Mol. Cell. Biol. 26,
22152225.
Thvenod, F., 2009. Cadmium and cellular signaling cascades: to be or not to be?
Toxicol. Appl. Pharmacol. 238, 221239.
Vandenabeele, P., Vanden Berghe, T., Festjens, N., 2006. Caspase inhibitors promote
alternative cell death pathways. Sci. STKE 2006, pe44.
Viau, M., Gastaldo, J., Bencokova, Z., Joubert, A., Foray, N., 2008. Cadmium inhibits
non-homologous end-joining and over-activates the MRE11-dependent repair
pathway. Mutat. Res. 654, 1321.
Wada, T., Penninger, J.M., 2004. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23, 28382849.
Whiteside, J.R., Box, C.L., McMillan, T.J., Allinson, S.L., 2010. Cadmium and copper
inhibit both DNA repair activities of polynucleotide kinase. DNA Repair (Amst.)
9, 8389.
Xiang, L.X., Shao, J.Z., 2003. Role of intracellular Ca2+ , reactive oxygen species,
mitochondria transmembrane potential, and antioxidant enzymes in heavy
metal-induced apoptosis in sh cells. Bull. Environ. Contam. Toxicol. 71,
114122.
Xu, Y., Kim, S.O., Li, Y., Han, J., 2006. Autophagy contributes to caspase-independent
macrophage cell death. J. Biol. Chem. 281, 1917919187.
Yang, P.M., Chen, H.C., Tsai, J.S., Lin, L.Y., 2007. Cadmium induces Ca2+ -dependent
necrotic cell death through calpain-triggered mitochondrial depolarization and
reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity.
Chem. Res. Toxicol. 20, 406415.
Zong, W.X., Ditsworth, D., Bauer, D.E., Wang, Z.Q., Thompson, C.B., 2004. Alkylating
DNA damage stimulates a regulated form of necrotic cell death. Genes Dev. 18,
12721282.