Cytotoxicity of diatom-derived oxylipins in organisms belonging to different phyla
1 Max-Planck Institute, Hans-Knöll-Str. 8, D-07745 Jena, Germany, 2 Station Biologique, CNRS, Mer et Santé (FRE 2775), INSU, UPMC, PO Box 74, 29682 Roscoff, France and 3 Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany
* Author for correspondence (e-mail: poulet{at}sb-roscoff.fr)
Accepted 20 May 2004
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Summary |
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Key words: diatom, oxylipin, cell toxicity, marine, non-marine organism, unsaturated aldehyde
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Introduction |
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,ß,
,
-unsaturated aldehydes are synthesised by
numerous organisms including mammals and higher plants
(Glasgow et al., 1986
;
Salch et al., 1995
).
Subsequently, this compound class was also detected in planktonic algae.
Wendel and Jüttner (1996
)
first detected 2E,4Z-decadienal in the freshwater diatom
Melosira varians (Bacillariophyceae). A series of molecules with this
structural element was subsequently found in marine diatoms
(Miralto et al., 1999a
;
Pohnert and Boland, 2002
;
d'Ippolito et al., 2002
,
2003
;
Pohnert et al., 2002
;
Adolph et al., 2003
), but not
all diatoms have the capability for aldehyde production
(Pohnert et al., 2002
). More
recently, other freshwater microalga belonging to the Chrysophytes and
Synurophytes were added to the list of producers of unsaturated aldehydes
(Watson and Satchwill, 2003
).
Several diatom species have developed the ability to synthesise these noxious
compounds by lipoxygenase-mediated transformation of polyunsaturated fatty
acids (Pohnert and Boland,
2002
). The production of reactive oxylipins is initiated upon cell
disruption such as would occur during mesozooplankton grazing
(Pohnert, 2000
). Aldehyde
formation is initiated by the release of polyunsaturated fatty acids, which
are further metabolized via a lipoxygenase/hydroperoxide lyase
pathway (Pohnert, 2000
,
2002
;
Pohnert and Boland, 2002
). The
deleterious effect of unsaturated aldehydes from diatoms on marine organisms
was first identified in copepods (Miralto
et al., 1999a
). When ingested by spawning females, these aldehydes
are responsible for a suite of physiological dysfunctions during egg
development, hatching and morphogenesis in larvae
(Miralto et al., 1999a
;
Romano et al., 2003
;
Poulet et al., 2003
;
Ianora et al., 2004
). In fact,
depending on the quantity of diatoms ingested by females
(Chaudron et al., 1996
), the
number of normal embryos and offspring is low or survivors do not reach
adulthood (Carotenuto et al.,
2002
; Poulet et al.,
2003
; Ianora et al.,
2004
). These molecules affect hatching and embryogenesis, two key
reproductive processes in copepods, and have therefore been classified as
antiproliferative oxylipins.
Following the work initiated by Poulet et al.
(1994) and Miralto et al.
(1999a
), which focused on the
diatom-copepod interactions, several other types of marine organisms have been
recently tested. Up until now, the noxious effects of unsaturated aldehydes
have been reported against a marine diatom (R. Casotti, S. Mazza, A. Ianora
and A. Miralto, unpublished) and for several marine invertebrates, including
ascidians (Tosti et al.,
2003
), tunicates (Miralto et
al., 1999b
), echinoderms
(Caldwell et al., 2002
;
Adolph et al., 2003
;
Romano et al., 2003
),
polychaetes (Caldwell et al.,
2002
) and crustaceans (copepods -
Romano et al., 2003
;
Poulet et al., 2003
;
Artemia - Caldwell et al.,
2003
). The majority of previous bioassays, conducted in
vitro with eggs and embryos, were related to the noxious effects of
2E,4E-decadienal on the embryonic and larval development of
these organisms. Although biological activity was observed in all cases, the
inhibitory mechanism still remains unknown.
The results presented here show that the unsaturated aldehyde
2E,4Z-decadienal, taken as a representative of the noxious
oxylipins synthesised by marine diatoms, could trigger several categories of
cell inhibition in a wide range of marine and non-marine organisms belonging
to different phyla. Observations were compared with structurally related
diatom-derived unsaturated aldehydes and the saturated aldehyde decanal, which
lacks the reactive Michael-acceptor element and which exhibited low activity
in sea urchin egg cleavage assays (Adolph
et al., 2003). Our main objective was to explore which cellular
pre-requirements have to be present for activity and what key physiological
functions could be inhibited by unsaturated aldehydes, using a range of
cellular models. This study suggests that diatom-derived oxylipins trigger
in vitro the inhibition of different cell processes, among which
apoptosis is probably the ultimate symptom. Findings are discussed in the
context of selecting key organisms as future tools to permit the further
elucidation of the inhibitory mechanism(s) involved at cellular and molecular
levels occurring in marine organisms naturally exposed to toxic diatom
diets.
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Materials and methods |
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Sub-samples of stock solutions of both unsaturated and saturated aldehydes,
diluted in dimethyl sulphoxide (DMSO; Sigma; 10 mg ml-1), were used
for the assays. Control experiments with DMSO and seawater were performed in
parallel. 2E,4E-decadienal,
2E,4E-octadienal, 5E,7E-9-oxononadienoic
acid and 4Z-decenal were tested against Bacillus megaterium,
Escherichia coli, Microbotryum violaceum, Mycotypha microspora, Dendryphiella
salina, Fusarium oxysporum, Asteromyces cruciatus and Chlorella
fusca. 2E, 4E-decadienal, 2E-decenal,
4Z-decenal and decanal were tested against the fungus
Saccharomyces cerevisiae. 2E,4E- decadienal and
decanal were tested with the bacterium Vibrio splendidus, the sea
urchin Sphaerechinus granularis and the oyster Crassostrea
gigas. With the copepod Calanus helgolandicus, the combined
effects of 2E,4E/Z,7Z-decatrienal,
2E,4E/Z-decadienal,
2E,4E/Z-octadienal,
2E,4E/Z,7-octatrienal,
2E,4E/Z-heptadienal and tridecanal were determined
with the diatom Thalassiosira rotula, which produces these
metabolites upon wounding (Pohnert et al.,
2002), and were tested through feeding experiments. A wide
concentration range (0-2x106 cells vial-1) of
diatoms was used in the diets.
Bacteria, fungi and algae
Test organisms were the bacteria Bacillus megaterium de Bary (Gram
positive), Escherichia coli (Migula) Castellani and Chambers (Gram
negative) and Vibrio splendidus biovar I ATCC 33125 (Gram negative),
the fungi Microbotryum violaceum (Pers.) Roussel (Ustomycetes),
Mycotypha microspora Fenner (Zygomycetes) and Fusarium
oxysporum Schltdl. (mitosporic fungi), the marine fungi Dendryphiella
salina (Sutherland) Pugh and Nigot and Asteromyces cruciatus
Moreau and Moreau ex Hennebert (Ascomycetes), the budding yeast
Saccharomyces cerevisiae (Ascomycetes), and the alga Chlorella
fusca Shih Krauss (Chlorophyceae).
Biological activity of aldehydes against microorganisms was tested in agar
diffusion assays. 50 µl of 1 mg ml-1 solutions of the test
compounds in DMSO/water (1:10) were pipetted onto sterile filter disks
(Schleicher & Schuell, Dassel, Germany). These were placed onto an
appropriate agar medium sprayed with a suspension of the test organisms:
Bacillus megaterium, Escherichia coli, Microbotryum violaceum, Mycotypha
microspora, Fusarium oxysporum, Dendryphiella salina, Asteromyces
cruciatus and Chlorella fusca. DMSO was used as control. Growth
media, preparation of test organism suspensions and incubation conditions are
described elsewhere (Schulz et al.,
1995). Agar diffusion assays were carried out according to Schulz
et al. (1995
). The radii of
the resultant inhibition zones were measured from the edge of the filter disks
and reported in millimetres.
Strains of the marine bacterium Vibrio splendidus were obtained
from Institut Pasteur, Lille, France, kept on Marine agar 2216 (DIFCO,
Franklin Lakes, NJ, USA) and grown in a liquid medium (Mueller-Hinton broth
added with sodium chloride 2%) for 24 h at room temperature, diluted with
sterile filtered seawater up to a cell density of 2x107 cells
ml-1. Inoculums were then spread on solid agar medium in Petri
dishes (Mueller-Hinton agar added with sodium chloride 15 g ml-1).
Plate 1 received five sterile disks, 6 mm in diameter (Schleicher &
Schuell): two antibiotic disks (Oxoid, Basingstoke, UK; 15 µg
disk-1 gentamycin; 30 µg disk-1 chloramphenicol), one
control disk (20 µl disk-1 DMSO), one with
2E,4E-decadienal (6.6 µg disk-1) and one with
decanal (6.6 µg disk-1). Plate 2 received six disks: one
antibiotic disk (15 µg disk-1 gentamycin), two with decanal (66
µg disk-1 and 6.6 µg disk-1) and three with
2E,4E-decadienal (33.3 µg disk-1, 6.6 µg
disk-1 and 0.66 µg disk-1). Plates were incubated in
duplicate at room temperature for 24 h. Thediameters of the resultant
inhibition zones were measured and reported in millimetres. This antibiotic
susceptibility test (Bauer et al.,
1966) was used to establish the bacteriostatic activity of
2E,4E-decadienal on V. splendidus cultures, in
comparison to decanal, DMSO as a negative control and antibiotics as positive
controls.
Budding yeast
The budding yeast strains used in this study were as follows. Wild-type
(WT) strain: Mata, ade1-14, trp1-289, his3200, ura3-52,
leu2-3,112 (strong strain of 74-D694), described by Chernoff et al.
(1995
). STRg6 strain:
Mata, erg6:TP/, ade1-14, trp1-289, his3
200, ura3-52,
leu2-3,112, described by Bach et al.
(2003
). A sample of an
overnight culture of either erg6
mutant (STRg6 strain) or the
corresponding wild-type strain was spread on a Petri dish containing YPD
(yeast extract peptone dextrose medium)-rich medium and small filters
(Schleicher & Schuell) placed on the agar surface. Individual compounds
were applied to each filter (20 µl of a 3 mmol l-1 solution in
DMSO). DMSO, the compound vehicle, was used as a negative control.
Bioassays with invertebrates
Echinoderm
Sea urchins, Sphaerechinus granularis (Lamarck), collected along
the Brittany coast (France), were transported in seawater containers to the
Marine Station within 3 h, where they were kept in running seawater. Male and
female gametes were collected and fertilisation was conducted in
vitro, following the protocol described by Meijer et al.
(1991). Dense sample solutions
(100 µl, with 15 000-20 000 embryos ml-1) of newly fertilised
eggs (5-9-min-old embryos) were placed in 900 µl filtered seawater in 5 ml
culture wells (Nunc, Roskilde, Denmark) enriched with increasing
concentrations of test aldehydes, in the range of 1-250 µmol l-1
(final concentration in well), at a constant temperature of 20°C.
Observation of the proportions of first (two blastomeres) and second (four
blastomeres) cell cleavages was performed for each compound 2-3 h later, in
replicate samples of 100 embryos each, and compared with controls (embryos
incubated in filtered seawater and with DMSO, 2.5% per volume seawater). This
protocol was used to evaluate the effect of aldehydes on cell division during
the early phase of embryonic development.
Mollusc
Oysters, Crassostrea gigas (Thumberg) (60-70 g wet mass each),
purchased from an oyster farmer in the Bay of Morlaix (France), were
maintained undisturbed for a 7-day acclimation period in tanks (50 oysters per
tank) containing 110 litres of aerated and continuously flowing natural
seawater (50 l h-1) at 15-16°C in the laboratory. For all
tests, individual oysters were taken from the tank prior to sampling
haemolymph. The right side of the shell in each oyster was notched, allowing
the sampling of blood in the adductor muscle using 2 ml syringes and 26 gauge
x 1.3 cm needles. The rapidity of the procedure (1-2 min per sample)
ensured that the effect of sampling on stress-induced catecholamine hormone
release was minimal (Lacoste et al.,
2001a). Haemolymph samples (0.5-1 ml) were pooled from 5-6 oysters
in tubes kept on ice. Haemocyte concentration was determined with a
haemocytometer and adjusted to 106 cells ml-1 by the
addition of modified Hanks' balanced salt solution (MHBSS), consisting of HBSS
adjusted to ambient seawater salinity (35 p.p.m.) and containing 2 g
l-1 D-glucose
(Anderson et al., 1994
), in
order to prevent cell aggregation. For each category of bioassays, new blood
solution samples were prepared.
Four bioassay categories, with concentrations ranging between 2 and 50
µmol l-1, were performed: (1) observation of cytoskeleton
structure, (2) apoptosis induction, (3) phagocytosis and (4) haemocyte
oxidative burst response. In assays related to cytoskeleton, apoptosis and
phagocytosis, all blood samples enriched with 10 µl of DMSO (controls) or
aldehydes were incubated in a humidified chamber in the dark at 20°C for
30 min in order to allow haemocytes to adhere on the plate and cell
penetration of test compounds. Detailed protocols used with these assays are
described by Panara et al.
(1996) and by Lacoste et al.
(2001a
,b
,c
,
2002
). These assays were based
on the evaluation of the multiple responses of oyster haemocytes to aldehydes,
in terms of proportions (%) of abnormal cytoskeleton, apoptotic and phagocytic
cells, in comparison with controls incubated in DMSO and filtered seawater.
The cytoskeleton was observed in rhodamine-phalloidin-stained oyster
haemocytes (Sigma) (Panara et al.,
1996
). Cells entering apoptosis were revealed with fluorescein
isothiocyanate (FITC)-Annexin V (Sigma) double labelling with propidium iodide
(Sigma) (Bossy-Wetzel and Green,
2000
; Lacoste et al.,
2002
). Phagocytosis was monitored with blood samples incubated on
glass slides, following a protocol described by Lacoste et al.
(2001b
). At the end of the
incubation period, 10 µl of 0.95 µm green fluorescent latex microspheres
(Polysciences Europe, Epplheim, Germany) were added to the sample to obtain a
ratio of 10 microspheres cell-1. Haemocytes were further incubated
for 30 min to allow phagocytosis to occur. In all assays, blood cell samples
were fixed at the end of the incubation periods with 3.7% formaldehyde for 15
min and observed under an Olympus BX 61 epifluorescent microscope or under an
IX Fluoview confocal microscope equipped with argon-krypton lasers. For
chemiluminescence assays, a protocol described by Lacoste et al.
(2001c
) was utilised. Zymosan
(Sigma) particles were used to stimulate the oyster haemocytes at a
concentration of 1 mg ml-1. The chemilumigenic probe used was
luminol (Sigma; 10-4 mol l-1 final concentration) added
to 1 ml cell suspensions containing 106 cells ml-1.
Baseline chemiluminescence was recorded 15 min before addition of Zymosan
particles (80 particles cell-1) and the luminescence response was
recorded using a Lumat LB 9507 luminometer (EG&G Berthold, Pforzheim,
Germany) every 3 min for 60 min. Chemiluminescence counts [relative light
units per second (RLU s-1)] for each tested compound were plotted
against time. All four series of assays were repeated three times each.
Crustacea
Copepods, Calanus helgolandicus (Claus), were collected offshore
from Roscoff (France) and transported within 2 h to the Marine Station.
Batches of 30 sexually mature females were sorted and acclimated individually
in containers filled with 100 ml filtered seawater (0.22 µm) for 24 h at
17°C. At the end of this initial period, females were fed with a diatom
culture in the exponential phase of growth (Thalassiosira rotula,
strain CCMP 1647) known to produce several unsaturated aldehydes
(Pohnert et al., 2002). The
algal culture conditions were similar to those described by Pohnert et al.
(2002
). In each incubator, 20
ml of dense diatom cultures were added to 80 ml filtered seawater. After
dilution of cultures, concentration of diatoms in diets ranged from
2x103 to 2x106 cells ml-1 in
vials. Diet was renewed daily during an 8-day period. The concentrations of
noxious aldehydes potentially available in the diets were measured
(Pohnert et al., 2002
; T.
Wichard et al., unpublished) in a test T. rotula culture during the
stationary phase of growth. This diatom can produce
2E,4E/Z-isomeric mixtures of
2,4,7Z-decatrienal and octadienal (not exceeding 2 fmol
cell-1 combined: this value was used to extrapolate potential
oxylipin values in Fig. 8A) and
minor amounts of 2,4-heptadienal, 2,4,7-octatrienal and
2E,4E-decadienal upon wounding. Feeding rates were not
evaluated; however, ingestion of diatoms by C. helgolandicus females
was estimated indirectly by counting faecal pellet production, which varied
from 6 to 30 faeces female-1 day-1, increasing with cell
density (Chaudron et al.,
1996
). The daily production and hatching success of eggs were
monitored following a protocol described by Laabir et al.
(1995
). Eggs in control
treatments were obtained following the same protocols, except that females
were fed a culture (104 cells ml-1) of the
dinoflagellate Prorocentrum minimum, which is unable to synthesise
the unsaturated aldehydes in question
(Pohnert et al., 2002
). This
protocol was used to evaluate, in vivo, the effect of diatom-derived
unsaturated aldehydes on cell division during embryogenesis in C.
helgolandicus compared with dinoflagellate control.
|
Observations of normal and abnormal embryonic cell division were performed
in samples stained with Hoechst 33342 (Sigma) specific for DNA, following the
protocol described by Poulet et al.
(1995). Samples were observed
in fluorescent light with an Olympus microscope. Nauplius larvae, which
hatched and survived the maternal-food effect during the incubation period
with diatoms, were collected beyond incubation day 3-5. This is the minimum
delay required to observe cell anomalies in Calanus embryos induced
by toxic diatoms ingested by spawning females
(Poulet et al., 1995
). A
double labelling method, FITC-Annexin V + propidium iodide, was used to
diagnose cell degradation processes in N1 stage nauplii, following a protocol
described by Poulet et al.
(2003
). Observation and
estimate of the proportions (%) of nauplii (12-20 larvae per sample),
intoxicated by maternal diatom diets and presenting apoptotic cell
degradations, were compared with controls using the same confocal Olympus
epifluorescent microscope.
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Results |
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Results with the marine bacterium Vibrio splendidus are presented
in Fig. 1. The diameter of the
inhibition zone was high with antibiotics (15 µg disk-1 bis
gentamycin, 21 mm; 30 µg disk-1 chloramphenicol, 36 mm) used as
references. Growth inhibition was 14 mm with the 33.3 µg disk-1
2E,4E-decadienal and lower (9 mm) at concentrations of
6.6 µg disk-1 and 0.66 µg disk-1. Elevated
concentrations of decanal (66.6 µg disk-1) resulted in slight
growth inhibition. DMSO did not affect bacterial growth significantly. From
these results and according to the chart of zone sizes
(Bauer et al., 1966
;
12
mm), we concluded that V. splendidus was sensitive to
2E,4E-decadienal in a concentration range comparable with
established antibiotics but insensitive to decanal.
|
Budding yeast
The wild-type (WT) Saccharomyces cerevisiae strain was found to be
insensitive to 9.1 µg disk-1 2E-decenal,
2E,4E-decadienal, 4Z-decenal and decanal, as
indicated by the absence of a growth inhibition halo where these compounds
were spotted (Fig. 2).
Interestingly, a 9.1 µg disk-1 of the Michael-acceptors
2E-decenal and 2E,4E-decadienal was found to
significantly inhibit cell growth of the STRg6 strain, whereas
4Z-decenal and decanal, the non-conjugated aldehydes of comparable
chain length, did not result in growth inhibition compared with DMSO
(Fig. 2). STRg6 strain lacks
the ERG6 gene. Such a deletion is known to increase cell
permeability, probably due to an increase in cell wall and/or plasma membrane
permeability (Blondel et al.,
2000). Indeed, wild-type budding yeast cells are slightly
permeable to a number of drugs, probably because, in addition to a plasma
membrane, they also have a cell wall. Deletion of the ERG6 gene,
which is involved in ergosterol biosynthesis, is one of the genetic ways to
increase cell permeability. The fact that erg6
cells were
sensitive to 2E-decenal and 2E,4E-decadienal (as
compared with WT cells) suggested that the cell wall and plasma membrane are
involved in the resistance against these molecules and, furthermore, that, in
order to be toxic, the inhibiting compounds must enter the cell or be in
direct contact with the plasma membrane.
|
Echinoderm
Second embryonic cell cleavage in Sphaerechinus granularis
occurred normally with DMSO in controls and elevated concentrations of decanal
(>80 µmol l-1). With <10 µmol l-1
2E,4E-decadienal, cells did cleave normally. At 10 µmol
l-1, cell cleavage was blocked in >50% of embryos and reached
100% at concentrations above 20 µmol l-1
(Fig. 3A;
Adolph et al., 2003).
Observations of 2-3 h-old embryos revealed the normal four blastomeres in
samples treated with DMSO, <80 µmol l-1 decanal or <10
µmol l-1 2E,4E-decadienal
(Fig. 3B1). Cell division was
blocked in samples assayed with 2E,4E-decadienal at
concentrations of >15 µmol l-1
(Fig. 3B2), whereas impairment
of development by elevated concentrations of decanal (>80 µmol
l-1) was induced by a subsidiary toxic effect, as revealed by small
spheres next to the egg membrane (Fig.
3B3).
|
Oyster
Results in Figs 4,
5,
6,
7 show the multiple inhibitory
effects of 2E,4E-decadienal (2-50 µmol l-1) on
the structure and key physiological functions of oyster haemocytes. The
results are compared with the effects of decanal, DMSO and filtered
seawater.
|
|
|
|
The haemocyte cytoskeleton was affected to different intensities by all
treatments (Fig. 4A). The
shapes of the cytoskeleton of disturbed and non-disturbed haemocytes are
compared in Fig. 4B. Cytoskeleton was well extended in normal cells
(Fig. 4A1) whereas it presented
a compact, spherical shape in abnormal cells
(Fig. 4A2). The background
inhibition induced by the handling of cells in seawater and in DMSO was
27-30%. In the presence of decanal, 41-46% of haemocytes also presented
abnormal spherical shapes. With added 2E,4E-decadienal,
43-59% of cells were affected. This effect was dose dependent and
significantly higher than with decanal (Student's t-test,
N=100, =0.05). With both aldehydes, shape anomaly of the
cytoskeleton was 12-28% above the DMSO control (significant Student's
t-test, N1, N2 and
N3=100 cells each,
=0.05).
Apoptotic haemocytes, detected with the FITC-Annexin V + propidium iodide
double labelling method, were observed in all treatments
(Fig. 5). Pictures of normal
(propidium positive, annexin negative; nucleus in red) and abnormal (propidium
positive, annexin positive: nucleus in red and cell membrane in green)
haemocytes are shown in Fig.
5B. Proportions of abnormal cells in seawater and DMSO controls
were 32 and 39%, respectively. Proportions of apoptotic cells in samples
treated with the respective aldehydes were dose dependent, ranging between 45
and 75%, which was significantly higher than in controls
(Fig. 5A; Student's
t-test, N1 and N2=100 cells
in each control and test sample, =0.05). Highest proportions of
apoptotic cells occurred in samples treated with
2E,4E-decadienal, which were 10-20% above decanal, 17-36%
above DMSO and 25-45% above seawater backgrounds.
Results on the phagocytosis bioassays are shown in
Fig. 6. With DMSO, inhibition
of phagocytosis in haemocytes was low: 3% above background level measured
with seawater. With decanal, inhibition values of 2 and 9% (2 µmol
l-1 and 50 µmol l-1 decanal) were not significantly
different from controls. With 2E,4E-decadienal, inhibition
was also dose dependent. Values, corresponding to 6 and 18% (2 µmol
l-1 and 50 µmol l-1 decadienal), were significantly
higher than those measured with both DMSO and decanal
(Fig. 6A;Student's
t-test, N1 and N2=100 cells
in each control and test sample,
=0.05). Pictures of three optical
sections of haemocytes observed with the confocal microscope, with
(phagocytosis) and without (blocked phagocytosis) fluorescent green
microspheres inside the cells (see green spot P in
Fig. 6B1), reflecting active
and non-active cells blocked by decadienal, respectively, are shown in
Fig. 6B1,2.
Fig. 7 shows the
chemiluminescence responses of oyster haemocytes, assayed with seawater, DMSO,
decanal and 2E,4E-decadienal before and after addition of
Zymosan. The background levels of the oxidative burst measured in assays were,
on average, below 800 RLU s-1 in all samples before addition of
Zymosan. The maximum oxidative-burst response of haemocytes, with peaks
detected 15 min after addition of Zymosan, reflected the inhibition level of
each chemical treatment. In seawater controls, the mean oxidative burst was
7000 RLU s-1. With DMSO, it was slightly above 5000 RLU
s-1, corresponding to 21-24% inhibition compared with controls.
With decanal and 2E,4E-decadienal, the dose-dependent
responses were below 3200 RLU s-1. At the same concentration (2 or
50 µmol l-1), inhibition exerted by decadienal was significantly
higher than that by decanal and chemiluminescence was 63-73% below values in
seawater controls (Student's t-test, N=16,
=0.05).
Copepod
Results with Calanus helgolandicus are reported in
Fig. 8. Eggs spawned by females
fed the dinoflagellate Prorocentrum minimum (control diet;
unsaturated-aldehyde free) underwent 100% normal cell division during
embryonic development (Fig.
8A,B1). In these series, embryos collected during the 8-day
incubation period hatched normally, giving rise to normal N1 stage larvae
(Fig. 8C1,2). With females fed
the diatom Thalassiosira rotula, the proportion of eggs presenting
abnormal cell division increased beyond day 3 in relation to diatom
concentrations in diets (Fig.
8A). T. rotula produces reactive aldehydes upon wounding.
The potential availability of these unsaturated aldehydes via a
natural diet was measured on one occasion (104 cells
ml-1) and extrapolated to the other diatom cell concentrations in
vials (Fig. 8A; T. Wichard, S.
Poulet and G. Pohnert, unpublished). In this case, the majority of embryos
collected on day 4 showed abnormal cell divisions
(Fig. 8B2,3). The few hatched
larvae expressed apoptotic cell degradations inside their bodies (green
spots), as revealed by the FITC-Annexin V double labelling method
(Fig. 8C3).
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Discussion |
---|
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---|
Depending on the organism, inhibitions triggered by
,ß,
,
-unsaturated aldehydes concerned either cell
proliferation, cell division, structure of cytoskeleton, phagocytosis, the
inhibition of the induction of an oxidative burst, or apoptotic cell
degradations (Figs 1,
2,
3,
4,
5,
6,
7,
8). Previous results by
Bisignano et al. (2001
) have
shown that unsaturated aldehydes have a broad antimicrobial spectrum. Our
results suggest that these oxylipins can affect various key physiological cell
processes in relation to their non-specific chemical affinities for
biomolecules (Esterbauer et al.,
1975
; Spiteller,
2001
; Luczaj and Skrzydlewska,
2003
). They reveal, for the first time, that inhibition of cell
proliferation by
,ß,
,
-unsaturated aldehydes,
determined in marine and non-marine organisms, is conserved from bacteria to
crustaceans. The fact that 2E,4E-decadienal and
2E-decenal were active in both erg6
cells of
Saccharomyces cerevisiae and other tested organisms (Vibrio
splendidus, Sphaerechinus granularis, Crassostrea gigas and Calanus
helgolandicus) suggests that, among the biochemical pathways targeted by
the reactive aldehydes, at least those involved in the cell proliferation
process might be evolutionarily conserved.
Among prokaryotes and eukaryotes tested in the present paper, the fungi
Dendryphiella salina, Fusarium oxysporum, Asteromyces cruciatus and
Saccharomyces cerevisiae (WT strain) and the bacteria Bacillus
megaterium and Escherichia coli were not significantly affected
by unsaturated nor saturated aldehydes under our experimental conditions. Cell
proliferation was weakly inhibited by 2E,4E-decadienal in
the terrestrial fungus Mycotypha microspora and the alga
Chlorella fusca and strongly inhibited in the marine bacterium
Vibrio splendidus and the genetically modified yeast
Saccharomyces cerevisiae (strain STRg6)
(Fig. 2). Comparison of results
obtained for WT and STRg6 strains of the budding yeast, Saccharomyces
cerevisiae, strongly suggests that in this eukaryotic species, the
chemical resistance to ,ß,
,
-unsaturated aldehydes is
related to cell wall and/or plasma membrane impermeability. WT and STRg6
strains are only distinguished by the presence or absence of the ERG6
gene, which results in increased cell permeability in the mutant
(Blondel et al., 2000
). This
direct comparison suggests that the
,ß,
,
-unsaturated
aldehydes can cause unspecific damage when they reach the inside of the cells.
The reduced cell permeability might thus be the reason for resistance in the
insensitive organisms tested here.
Cells belonging to marine invertebrates in our assays all responded to
unsaturated aldehydes. Many of these marine invertebrates rely on diatom diets
and are therefore exposed to diatom-derived oxylipins. This includes
crustaceans (copepods; Fig. 8),
molluscs (oysters; Fig. 7),
echinoderms (sea urchins; Fig.
3) and annelids (Caldwell et
al., 2002). Activity of 2E,4E-decadienal was
reported earlier in both marine invertebrates
(Tosti et al., 2003
;
Caldwell et al., 2002
) and
non-marine vertebrates, such as human cell lines
(Nappez et al., 1996
;
Miralto et al., 1999a
;
Spiteller, 2001
). That the
observed effects are not only found in vitro is demonstrated by the
female incubation experiments (Fig.
8) where the concentration-dependent effect of diatom diets on
copepods is shown. The aldehyde-producing diatom T. rotula, as a
diet, clearly reduced the hatching success of copepod eggs in a
concentration-dependant way, compared with incubation experiments where C.
helgolandicus was fed either P. minimum
(Fig. 8A) or T. rotula
(strain CCMP 1018), diets that do not produce any of the unsaturated aldehydes
(Pohnert et al., 2002
). These
results indicate that unsaturated aldehydes, potentially available in diets
and ingested by spawning females, are presumably responsible in vivo
for mitotic cell dysfunction in embryos and apoptotic cell degradations in the
newborn larvae, following a dose-dependent response.
Inhibition of cell division, apoptotic and necrotic cell degradations by
2E,4E-decadienal have already been reported in experiments
achieved in vitro and in vivo
(Caldwell et al., 2002;
Romano et al., 2003
;
Poulet et al., 2003
;
Ianora et al., 2004
). Recent
results (Romano et al., 2003
)
have suggested that decadienal induces caspase-independent apoptosis in
copepod embryos, whereas decadienal-mediated apoptosis in sea urchin embryos
was caspase dependent. Once again, these results reflect the non-specific
activity of decadienal, which has a potential reactivity against DNA, enzymes,
peptides, neuro-transmitters, hormones and other cell-signalling molecules
involved in different key cell-signalling pathways. We suggest that once one
of these key pathways and corresponding physiological cell functions are
disturbed, damaged cells will enter apoptosis. In fact, present results (Figs
4,
5,
6,
7), focused on the oyster
haemocyte response, strengthen the hypothesis that decadienal non-specifically
targets several biochemical pathways and thus can induce a range of cell
disorders before cell death. Decadienal seems to be a strong inhibitor of
several physiological functions in oyster cells, since the noxious effects of
this aldehyde could be clearly distinguished from the toxic effects caused by
decanal (Figs 4,
5,
6,
7). This is also the case for
sea urchin embryos (Fig. 3B). As shown in oyster blood cells (Fig.
4B), disturbance of the cytoskeleton in sea urchin embryos by
toxic diatom extracts has been also reported by Buttino et al.
(1999
), who assumed that
depolymerisation of microtubules was involved in the blockage of tubulin
organization.
Significant progress has been recently achieved regarding the formation,
reactivity and toxicity of aldehydes originating from diatoms
(Pohnert, 2000;
Pohnert and Boland, 2002
;
Adolph et al., 2003
; d'Ippolito
et al., 2002
,
2003
;
Romano et al., 2003
). Although
apoptosis has been identified as one cause of cell death in copepods
(Romano et al., 2003
;
Poulet et al., 2003
), we
suspect based on our results that it may not be the first nor the unique cell
disorder triggered by these noxious compounds. In fact, previous reports with
the Michael-acceptor 4-hydroxynonenal have shown that many other processes
could be inhibited by this aldehyde before cells enter apoptosis
(Brambilla et al., 1986
;
Comporti, 1998
;
Buszczak and Cooley, 2000
; see
review by Comporti, 1998
).
This view is also supported for the
,ß,
,
-unsaturated
aldehydes tested with oyster haemocytes (Figs
4,
5,
6,
7). Together, our results have
identified the wide spectrum of cell symptoms related to diatom-oxylipin
toxicity. Despite our first steps in marine invertebrates, neither the
molecular targets nor the inhibitory mechanisms are clearly identified.
Clarifying these processes remains a challenge. Assuming that apoptotic and
necrotic cell degradations observed in copepod embryos
(Romano et al., 2003
) and
larvae (Fig. 8C3;
Poulet et al., 2003
) are
probably the ultimate phases of cell disorders, we still do not know the
timing and link between the cell symptoms, observed in the sea urchin, oyster
and copepod samples in response to the unsaturated aldehydes, or their
adducts. Clarifying the impact of aldehydes, in terms of cell location, adduct
formation, molecular targets and timing between the cell symptoms, would
greatly help our understanding of these puzzling antiproliferative principles
produced by diatoms. In fact, new assays are requested to clarify how
oxylipins deregulate cell homeostasis in marine invertebrates.
Although the antiproliferative activity of diatom-derived oxylipins was
first identified in copepods (Miralto et
al., 1999a), these crustaceans may not be the ideal tools for
elucidating cell mechanisms involved in the inhibitory process. Several
practical limitations are related to the low egg numbers and non-synchronous
cell divisions in successive batches spawned by females. As shown by Wonisch
et al. (1998
), the yeast
Saccharomyces cerevisiae could be one suitable tool for the
elucidation of the mode of action of reactive oxylipins, because the genome
has been sequenced and culturing of strain STRg6 is easy, which is not the
case with copepods, sea urchins or oysters. The identified erg6
mutant, which is susceptible to a broad range of diatom-derived oxylipins, can
provide a useful model system for further studies on inhibitory mechanisms on
the cellular level. Knowing that the antiproliferative model is conserved
among different phyla, we recommend usage of this genetically modified yeast
strain in future bioassays in order to further elucidate the molecular targets
and cell mechanisms involved in numerous marine invertebrates naturally
exposed to diatom-derived oxylipins.
Sailors from the Roscoff Marine Station are deeply thanked for collecting samples at sea. This work was partially funded by the European program EGIDE/PROCOPE, CNRS and DFG (G.P., T.W.). Prof. W. Boland is acknowledged for the support during the preparation of this work. We thank Ch. Cordevant, from Institut Pasteur, Lille, for providing the V. splendidus strain, and Dr P. Potin, from our Biological Station, for the use of his luminometer. Referees are deeply acknowledged for their helpful comments and suggestions.
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