UV incites diverse levels of DNA breaks in different cellular compartments of a branching coral species
National Institute of Oceanography, Israel Oceanographic and Limnological Research, Tel-Shikmona, PO Box 8030, Haifa 31080, Israel
* Author for correspondence (e-mail: buki{at}ocean.org.il)
Accepted 12 January 2005
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Summary |
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Key words: comet assay, coral, DNA breakage, DNA repair, free radicals, UV radiation
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Introduction |
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Solar radiation (including ultraviolet radiation, UVR, at 280400 nm
wave length) alone, or in combination with other factors, such as increased
seawater temperature, has been cited as a major cause of coral bleaching in
field and laboratory manipulations
(Siebeck, 1988;
Glynn, 1993
;
Shick et al., 1996
;
Brown, 1997
;
Hoegh-Guldberg, 1999
;
Anderson et al., 2001
;
Wissmann, 2003
;
Lesser and Farrell, 2004
).
However, attempts to correlate coral bleaching with specific wave bands (UVR
vs photosynthetically active radiation, PAR, at 400700
nm)proved to be insubstantial. For example, Gleason and Wellington
(1993
) reported results from
field experiments, showing that, irrespective of the water temperature, the
between-depths coral transplants bleached in response to UVR increase.
However, their results have since been questioned
(Dunne, 1994
) because their
experimental design had not taken into account the slight differences in PAR
between treatments, nor the possibility of an interactive effect between PAR
and UVR. Considerable numbers of investigations were further directed towards
the ecological and physiological consequences of solar irradiation on coral
reef photoautotrophic and other epifaunal organisms
(Jokiel, 1980
;
Glynn, 1993
;
Brown et al., 1995
;
Fitt and Warner, 1995
;
Le Tissier and Brown, 1996
;
Shick et al., 1996
;
Brown, 1997
;
Hoegh-Guldberg, 1999
;
Warner et al., 1999
;
Lesser, 2000
;
Lesser and Farrell, 2004
).
Therefore, while a fair amount is known about the cellular processes that lead
to loss of algal cells from coral tissue during bleaching
(Lesser et al., 1990
;
Gates et al., 1992
;
Brown et al., 1995
;
Le Tissier and Brown, 1996
;
Warner et al., 1999
;
Sawyer and Muscatine, 2001
;
and literature therein), very little is known about the impacts of UVB
radiation on the DNA level in hermatypic corals and the possible alignment of
elevated DNA damages with coral bleaching. Only a single study observed, in
solar simulation, the increase of thymine dimers in Porites colonies
exposed to artificial solar irradiance
(Anderson et al., 2001
) and
another study (Lesser and Farrell,
2004
) has evaluated the formation of cyclobutane pyrimidine
dimmers following exposure of coral colonies to UVR.
To determine the potential genotoxic impact of UVB radiation on
coralalgal symbiosis, we evaluated by in vitro experiments the
DNA damage inflicted on the three major cellular compartments of this
association, on the non-symbiotic fractions of coral and algal cells and the
symbion-entity (the holobiont, the in hospite unit). The coral
species used in this study is the Indo-Pacific, shallow water branching form,
Stylophora pistillata. UV-induced DNA damage and DNA repair levels
were evaluated by the single cell gel electrophoresis assay, also known as the
comet assay, one of the most reliable and sensitive methods for evaluating DNA
damage induced in individual cells by various agents
(Mitchelmore and Chipman,
1998; Avishai et al.,
2003
; Reinhardt et al.,
2003
).
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Materials and methods |
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UV irradiation
In each experiment, cells were divided into three 24-well plates (TPP,
Trasadinger, Switzerland), suspended in culture medium made of 2.5% Dulbecco
modified Eagle medium (DMEM) in 2x artificial seawater supplemented with
3% heat inactivated fetal calf serum (HI-FCS), 0.01 mol l1
HEPES, 2 mmol l1 glutamine and 1% of antibiotic cocktail (10
U ml1 penicillin G, 10 mg ml1
streptomycin, 25 µg ml1 amphotericin B). 2xASW was
prepared as the following: NaCl 13.67 g, KCl 0.412 g, CaCl2
2H2O 0.721 g, MgSO4 7H2O 5.57 g and
MgCl2 6H2O 3.05 g were added to 250 ml tissue culture
grade water. Cultures were incubated overnight (20 h) in the dark in a
humidified incubator (5% CO2), at 20°C. Irradiation was
performed in the dark by a UVB lamp (VL-6M, 16 W tube, a peak wavelength at
312 nm, power 12 W; Vilber Lourmat, Marne La Vallée, France) for 15, 30
and 45 min, respectively (equivalent to 4.05, 8.1 and 12.2 kJ
m2; irradiation was measured by a sensor, radiometer CUV3;
Kipp and Zonen, Delft, Holland). The spectral power distribution of the
broadband lamp was determined by measuring the integrated spectral irradiance,
since the sensor used does not record total lamp output. Lamp/specimen
geometry was kept identical in all experiments. After irradiation, cells were
collected by centrifugation (2400 g) at room temperature and
loaded onto glass slides for the comet assay. Repair experiments were left in
the dark for 1 h after irradiation.
The comet assay
In this assay, 10 µl of cell suspensions
(210x105 cells) were embedded in 90 µl of 0.65%
low-melting agarose (Amresco, Solon, OH, USA) layered on a Star-frost
microscope slide, precoated with 0.65% normal melting agarose. After 10 min of
solidification on ice, a third layer containing 120 µl of 0.65% low-melting
agarose was placed on top and left on ice for an additional 1015 min
until solidification. The cells were then lysed by immersing the slides for 1
h in a freshly prepared lysis solution (2.5 mol l1 NaCl, 100
mmol l1 EDTA, 10 mmol l1 Tris, 1% Triton
X-100, 10% DMSO, pH 10.0) at 4°C. After lysis, the slides were washed
three times (5 min each) in cold double distilled water and placed, for 20
min, in a horizontal gel electrophoresis apparatus containing freshly prepared
electrophoresis buffer (1.0 mmol l1 EDTA, 300 mmol
l1 NaOH, pH 13.0) to allow DNA unwinding. Electrophoresis
was done at 20 V (a starting current of 300 mA) for 20 min. Thereafter, the
slides were neutralized with three washes (5 min each) of 0.4 mol
l1 Tris, pH 7.5, dehydrated with ethanol, dried, stained
with 65 µl of 20 µg ml1 ethidium bromide solution and
viewed under a fluorescent microscope using a U-MNG filter (Olympus, Hamburg,
Germany). All steps were performed in the dark to prevent additional DNA
damage. The analysis was done on 400x magnification images. The cell
images were projected onto a high resolution Heper-HADTM (Sony, Tokyo,
Japan) CCD camera [8 bits (Applitec, Holon, Israel; LIS-700)] and analyzed
with Viscomet image analysis software using the MV Delta frame grabber (Matrix
Vision, Oppenweiler, Germany). DNA damage in each treatment was measured in
duplicates of 50 cells each, using two highly informative parameters
(Avishai et al., 2003): tail
extent (sum of all distances of each horizontal scan line from the first
signal pixel to the last signal pixel divided by number of scan lines) and
tail extent moment (tail length x percentage tail DNA). Controls
(algal-free cells, animal cells, holobionts) were handled as experimental
cells, but without UVR treatment.
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Results |
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Analysis of parameters studied (Fig. 2 depicts values for tail extent and tail extent moment) revealed clearly that under the three UVB doses used, the holobiont-entities of the tested colonies were significantly more sensitive to the irradiation than the other two cell types (P<0.05, using Duncan test; SPSS 10.0 for windows). Thus, not only was an increased DNA breakage distinct to this cell compartment (i.e. comet assay measurement of tail extent moment values, up to 5.0, 9.5 and 6.9 times higher than controls and for tail extent analyses up to 3.4, 4.6 and 4.1 times higher values than controls in the 4.05, 8.1 and 12.2 kJ m2 UVB doses, respectively; Fig. 2), but also DNA breakage levels in the holobiont fractions exceeded the levels observed for the other two compartments, the animal cells and the animal-free algal cell fractions, in most cases (P<0.05, Duncan test; up to 5.9, 9.9 and 13.4 times higher for 4.05, 8.1 and 12.2 kJ m2 doses, respectively; Table 1). Results of the lowest dose, 4.05 kJ m2 in experiment no. 2, were different. The holobiont entity is, therefore, more sensitive to the genotoxic impacts of UVB irradiation than the other cell compartments, the algal-free animal cells and algal cells.
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The damages observed may represent the combination of directly induced strand breaks, of alkaline-labile lesions and endonucleolythic incisions at the sites of base damage (mainly pyrimidine dimmers). Thus, not only the apparent levels of induced damage but also the efficiency and speed of base and nucleotide excision repair during the irradiation (which may differ between the compartments) may affect the results. Three other sets of irradiation experiments (30 min; 8.1 kJ m2) further revealed DNA repair patterns by comparing DNA breaks levels immediately after irradiation with those recorded after 1 h (under dark conditions) repair periods, in an attempt to elucidate possible different repair patterns in the three coral cellular compartments. As before (Fig. 2), the results (Fig. 3) documented a distinctive immediate increase in DNA damage levels in the holobiont cell compartment (P<0.05, Duncan test; up to 2.4 and 60.0 times higher than that recorded in the animal and algal cell fractions, respectively). One hour following the irradiation, in most of the animal and the algal-free cell fractions, a presumed active nucleotide and base excision repair mechanisms were reflected by increased DNA break levels (up to 4.7 for the animal cell compartment and up to twice for the algal cell compartment as compared with levels measured immediately after irradiation; Fig. 3). Conversely, 1 h after irradiation, the holobiont fraction demonstrated, in two out of the three experiments, a reduction in DNA breakage levels as compared with the levels recorded following the irradiation (down to 50%; Fig. 3). Two additional sets of experiments (data not shown) revealed similar results, namely, a 1 h post irradiation decrease in the holobiont DNA breaks values as compared with an elevated DNA breakage in most samples of the other cell compartments. The above results may suggest, therefore, that only in the holobiont fraction a rapid process of repair develops simultaneously to irradiation, without or with only minimal lag periods. This makes this cell fraction more vulnerable to synergistic impacts UV radiation and simultaneously activated other genotoxic agents (see below), a possibility that should be evaluated by additional sets of experiments on each individual cellular compartment.
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The role of UVR in bleaching seems to generate many disputes
(Brown, 1997;
Anderson et al., 2001
;
Douglas, 2003
). Some studies
(Helbling et al., 2001
)
maintain that natural levels of UVB are not sufficient to be acknowledged as
the major contributor to bleaching. Consequently, the genotoxic impact of UVR,
which was particularly addressed in phytoplanktonic organisms
(Gieskes and Buma, 1997
;
Helbling et al., 2001
) as a
consequence of the formation of cyclobutane pyrimidine dimers
(Mullenders and Berneburg,
2001
), was not implicated as a notable subject for bleaching in
symbiotic cnidarians. However, the discovery of the importance of solar UVR as
a factor affecting the biology of coral reefs is fairly recent
(Jokiel, 1980
;
Gleason and Wellington, 1993
).
Most studies concentrated on the impacts of physiological and biochemical
parameters, such as coral calcification, reproduction, amounts of UV-absorbing
compounds in coral tissues, body mass and photosynthesis capacities
(Siebeck, 1988
;
Gleason and Wellington, 1993
;
Glynn, 1993
;
Dunne, 1994
;
Brown et al., 1995
;
Le Tissier and Brown, 1996
;
Shick et al., 1996
;
Brown, 1997
;
Hoegh-Guldberg, 1999
;
Westholt et al., 1999
;
Wissmann, 2003
). Furthermore,
several studies (Lesser et al.,
1990
; Dykens et al.,
1992
; Lesser,
1996
; Lesser and Farrell,
2004
; Takahashi et al.,
2004
) considered the impacts of reactive oxygen species (ROS)
produced by UVR and the inherent susceptibility of symbiotic cnidarians to
oxidative stress, as playing major roles in coral bleaching. It is possible,
therefore, that the holobiont susceptibility to UVB radiation, as demonstrated
in this study, reflects a synergistic breakage of DNA strand augmented by the
formation of dimers between adjacent pyrimidines
(Anderson et al., 2001
), the
ROS damage (Kvam and Tyrrell,
1997
; Lesser and Farrell,
2004
) and fast repair mechanisms. Synergism between solar
radiation (that includes UVB radiation) and other environmental stressors,
like temperature, may also coalesce to produce stressful conditions
(Lesser et al., 1990
;
Glynn et al., 1992
;
Brown et al., 1995
;
Wissmann, 2003
;
Lesser and Farrell, 2004
),
including an increase in DNA damage.
This study reveals that natural levels of UVB radiation induce unalike DNA
breaks in different coral cells, a phenomenon that is followed by different
DNA repair rates. It is well documented that, in clear tropical seawater, UVR
penetrates to ecologically important depths
(Gleason and Wellington, 1993;
Shick et al., 1996
;
Brown, 1997
). Coral colonies at
1 m depth may receive up to 98% of surface UVB radiation
(Gattuso et al., 1991
). One
may also consider changes in UVR underwater attenuation, which may be
influenced by climate changes (Gleason and
Wellington, 1993
; Brown,
1997
). It is possible that short-term increases in UVR intensity,
under extremely calm (doldrums) clear water column conditions
(Gleason and Wellington, 1993
)
may contribute to bleaching in reef corals as a result of increased DNA
damage, specifically to the symbiont-entity. It is not clear yet as to what
extent the expression of antioxidant enzymes, one of the defense mechanisms of
symbiotic cnidarians against ROS (Richier
et al., 2003
), will be able to efficiently cope with this elevated
DNA damage, or what would be the consequences of faster repair mechanisms.
Anyhow, it is intriguing to find that the intimate coralalgal symbiotic
unit is strictly hypersensitive to UVB radiation, a point that should be
considered when discussing global changes and synergism between several
factors (Lesser et al., 1990
;
Gleason and Wellington, 1993
;
Glynn, 1993
;
Brown et al., 1995
;
Le Tissier and Brown, 1996
;
Shick et al., 1996
;
Brown, 1997
;
Hoegh-Guldberg, 1999
) that can
jointly result in massive bleaching of coral reefs worldwide. Such
consideration would help establishing the causative relationship between UVR
and apparent coral bleaching events based upon mechanistic rather than on
correlative information. Coral bleaching, then, is a considerably more
complicated mechanism phenomenon than portrayed in earlier studies
(Hoegh-Guldberg, 1999
).
Although there is as yet no experimented evidence to the claim that coral
bleaching is associated with DNA damage, the results of the present study
directly document the possible vulnerability of the holobiont entity to
elevated levels of DNA damages. This new approach of evaluating DNA damages
may contribute to our understanding and predicting the fate of coral reefs
under different scenarios of global change.
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Acknowledgments |
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