Exposure to ultraviolet radiation causes apoptosis in developing sea urchin embryos
Department of Zoology and Center for Marine Biology, University of New Hampshire, Durham, NH 03824, USA
* Author for correspondence (e-mail: mpl{at}cisunix.unh.edu)
Accepted 21 July 2003
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Summary |
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Key words: ultraviolet radiation, DNA damage, p53, apoptosis, oxidative stress, cell cycle, sea urchin, Strongylocentrotus droebachiensis
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Introduction |
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Surface waters of temperate near-shore coastal habitats (<10 m) contain
the planktonic life-history phases of many species of fish, macrophytes and
benthic invertebrates. Several species of echinoderms are important
broadcast-spawning members of benthic communities in the Gulf of Maine and may
therefore be susceptible to the detrimental effects of UVR. Exposure to UVR
affects fertilization success, the timing of cleavage and development time for
embryos and larvae of the green sea urchin Strongylocentrotus
droebachiensis (Adams and Shick,
1996,
2001
;
Lesser and Barry, 2003
). Many
of the embryos and larvae survive these exposures, but in the study by Lesser
and Barry (2003
) all
developmental stages tested exhibited significant DNA damage, measured as
cyclobutane pyrimidine dimers (CPDs), which was highly correlated with delays
in cell division and developmental delays. It was proposed that reactive
oxygen species (ROS), formed by the univalent reduction of molecular
O2, and including singlet oxygen (1O2),
superoxide radicals (O2-), hydrogen peroxide
(H2O2) and hydroxyl radicals (HO·),
formed via photodynamic action
(Asada and Takahashi, 1987
;
Fridovich, 1986
;
Halliwell and Gutteridge,
1999
; Valenzeno and Pooler,
1987
), act synergistically with the direct effects of UVR to cause
extensive DNA damage. This DNA damage leads to the expression of
characteristic markers of the cell cycle such as p53 and p21
that result in delays in cell division while DNA repair is taking place. DNA
damage, followed by p53 expression, has also been observed in the
embryos of the Atlantic cod (Lesser et
al., 2001
). If DNA repair is unsuccessful then those cells of the
developing embryo are slated for apoptosis or programmed cell death pathways.
Here, we provide evidence to support this hypothesis using embryos of the
green sea urchin S. droebachiensis. We show that DNA damage is caused
directly by exposure to UVR and provide evidence that DNA damage also occurred
indirectly via the production of ROS. In addition to this DNA damage
we observed differential expression of antioxidant and cell cycle genes and a
positive response to the TUNEL assay, which are consistent with the onset of
apoptosis in the cells of developing urchin embryos exposed to UVR.
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Materials and methods |
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Embryo experiments
Freshly fertilized embryos, blastula, and gastrula stages were exposed to
artificial visible radiation and UVR (290-700 nm wavelength) using four UV-340
lamps (Q-Panel, Cleveland, USA) and four F40 Sun lamps (General Electric)
suspended 15 cm from the top of the filters (see below) to provide a
downwelling mixed field (visible and UVR) exposure. The effects of UVR were
tested on embryos in 400 ml glass beakers at a density of 4-5 individuals
ml-1. Five treatments were used to partition the effects of UV-B
from UVA (320-400 nm) and visible radiation with three replicates per
treatment. WG and GG long-pass filters (Schott, Yonkers, USA)
(6''x6'') with nominal cutoffs (50% transmission) at 280, 305,
320, 375 and 400 nm were used to cover the beakers containing embryos. Embryos
were subjected to a 12.00 h:12.00 h light:dark cycle and experiments carried
out for 3 days. Using this design, successive replicate experiments were
carried out using embryos at different developmental stages for
Strongylocentrotus droebachiensis. At the end of 3 days all embryos
were collected for analyses as described below.
Measurements of ultraviolet radiation
For laboratory experiments UVR (UV-B and UV-A) and photosynthetically
active radiation (PAR, 400-700 nm) were measured using a wavelength- and
radiometrically calibrated [using National Institute of Standards and
Technology (NIST) traceable standards] CCD spectrometer with fiber optics
(Ocean Optics, Inc., Dunedin, USA). Three scans were taken and the mean values
(W m-2 nm-1) determined. Integrated values of unweighted
UVR (W m-2) were calculated for each treatment and biologically
weighted irradiances (W m-2) were obtained by multiplying the
unweighted irradiance by the DNA weighting function of Setlow
(1974).
Detection of DNA photoproducts using an enzyme-linked immunoabsorbent
assay (ELISA)
Cyclobutane pyrimidine dimer (CPD) formation was measured using the
procedures and monoclonal antibody (TDM-2) of Mori et al.
(1991). Genomic DNA was
isolated using commercially available kits (Easy-DNA, Invitrogen, Inc.,
Carlsbad, USA) and the quality and concentration determined
spectrophotometrically using 260/280 nm ratios. Subsequently, 50 ng of DNA
from each sample was used in an enzyme-linked immunoabsorbent assay (ELISA)
technique with TDM-2 as the primary antibody and an affinity-purified goat
anti-mouse IgG secondary antibody conjugated with horseradish peroxidase. The
final color development was read in flat-bottomed 96-well microtiter plates
using a plate reader (Bio-Rad, Inc., Hercules, USA) at 490 nm and the
absorbance units reported as described by Mori et al.
(1991
).
Western blots
From the experiment described above, protein extracts of individual embryos
(N=3) were homogenized using a tissue homogenizer in 10 mmol
l-1 Hepes buffer, pH 7.5, containing dithiothreitol (DTT) and
phenylmethylsulfonyl fluoride (PMSF) to prevent protein oxidation and certain
classes of protease activity, respectively. The homogenate was then
centrifuged at 500 g for 20 min and the supernatant saved for
analysis of protein (Bradford,
1976). Samples (N=3) from each treatment of equivalent
protein biomass were separated on SDS-PAGE gradient (4-15%) polyacrylamide
gels and then transferred to PVDF membranes (0.2 µm). The membrane was
blocked with 10% instant milk and immunoblotted using polyclonal antibodies
against cytosolic superoxide dismutase (SOD), a polyclonal antibody to human
p53 (23-mer; Kelly et al.,
2001
), p21 (Santa Cruz Biologicals, Santa Cruz, USA) and
cdc2 (p34, Santa Cruz Biologicals). The immunoblot was then developed
using a secondary antibody, at a titer of 1:2000, labeled with horseradish
peroxidase. Immunoblots of cdc2 were only performed on experiments with FFE
because of the lack of sufficient biomass in the other developmental stages.
The immunoblots were scanned and the optical density of the positive bands
measured using a calibrated gray scale and the gel-scanning procedures
described in NIH Image (version 1.61).
TUNEL assay
One of the diagnostic features of apoptosis is extensive damage to
chromatin and DNA cleavage that leads to DNA fragmentation via an
endogenous endonuclease. Identifying apoptotic cells can be accomplished using
an in situ enzymatic end-labeling technique known as the TUNEL
(TdT-mediated dUTP nick-end labeling) assay. Smears of experimental freshly
fertilized embryos were made on clean glass slides and allowed to air dry. The
cells were then fixed in buffered 4% paraformaldehyde for 1 h at 20°C.
Cells were permeabilized using 0.1% Triton X-100, 0.1% sodium citrate in
phosphate-buffered saline (PBS) at pH 7.4 and 4°C. Slides were rinsed in
PBS (x3) and the 3'-OH termini of the DNA strand breaks labeled
with modified nucleotides in an enzymatic reaction (In Situ Cell Death
Detection Kit, Fluorescein, No. 1 684 795; Roche, Palo Alto, USA). The
fluorescein-labeled DNA strand brakes in the nuclei of individual cells were
then observed and scored using epifluorescence microscopy. Slides were covered
with low fluorescence immersion oil and individual cells (N=200) from
each treatment group with distinct nuclear end labeling (green emission at
515-565 nm) were scored as positive cells.
Statistical analysis
CPD formation and the optical density of the positive western blots were
statistically analyzed using a one-way analysis of variance (ANOVA) at a
significance level of 5%. No unequal variances were detected using the
Fmax test, and individual treatment differences were
assessed using the Student-Newman-Keuls (SNK) multiple comparison test. Where
appropriate, ratios and percentages were arcsine- or log-transformed for
analysis and back-transformed for presentation. The TUNEL assay data were
scored as positive and negative cells and analyzed using the non-parametric,
multi-comparison Kruskal-Wallis test with Bonferroni/Dunn post-hoc
comparisons, with an adjusted significant P value of 0.005 to control
for experiment-wise Type I error.
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Results |
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Embryos of Strongylocentrotus droebachiensis exhibited significantly greater damage to DNA (ANOVA, P<0.001 on log-transformed values), measured as CPD formation (Fig. 1), at the end of the experimental UVR exposures. Multiple comparisons testing of the data from individual experiments at different embryonic stages showed that significantly more CPD formation occurred in the UV-B portion of the spectrum, while treatments without any UVR (GG 400 filter) always showed the lowest concentration of CPDs (SNK, P<0.05; Fig. 1).
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Western blots revealed a single band at 17 kDa, which corresponded to a Cu/Zn SOD standard (bovine erythrocytes; Sigma, Inc., St Louis, USA). Densitometer scans of SOD immunoblots showed that urchin embryos exposed to UVR expressed significantly greater concentrations of SOD (ANOVA, P<0.05) (Fig. 2A), with significant differences (multiple-comparison SNK, P<0.05) between the UV-B treatments (WG 280, WG 305 filters) and UV-A and control groups in most cases (Fig. 2A). Additionally, densitometer scans of immunoblots of the cell cycle genes p53 and p21 showed a similar significant (ANOVA, P<0.05) pattern of increasing p53 (53 kDa) and p21 (21 kDa) protein after exposure to UVR, especially the UV-B portion of the spectrum (SNK, P<0.05; Fig. 2B,C). The lone exception was the experiment on the gastrula stage, where p21 showed a non-significant trend of increasing protein concentration with increasing UVR (ANOVA, P=0.11). Lastly, for the FFE of Strongylocentrotus droebachiensis, the densitometer scans of immunoblots for cdc2 (p34) showed an inverse and significant (ANOVA, P<0.05) pattern of decreasing protein concentration after exposure to UVR (Fig. 2D).
|
Experimental FFE showed a significantly (Kruskal-Wallis test; P<0.001 when corrected for ties) greater number of TUNEL-positive cells observed with DNA strand breaks (Fig. 3F). Embryos exposed to the shortest wavelengths of UVB (280 nm, Fig. 3A) showed a greater percentage (36%) of TUNEL-positive cells than those in all other treatment groups (Bonferroni/Dunn test; P<0.005, 305 nm, 32%, Fig. 3B;320 nm, 18%, Fig. 3C; 375 nm, 17%, Fig. 3D; 400 nm, 10%, Fig. 3E). Blastula- and gastrula-stage embryos showed a distinct difference between UV-B and UV-A wavelengths; UV-B exposed cells contained significantly more TUNEL-positive cells (blastula: 280 nm, 78%; 305 nm, 57%; 320 nm, 53%; gastrula: 280 nm, 100%; 305 nm, 61%; 320 nm, 54%) than those exposed to longer wavelength UV-A (blastula: 375 nm, 37%; gastrula: 375 nm, 43%) or visible radiation only (blastula: 400 nm, 34%: gastrula: 400 nm, 29%) (Bonferroni/Dunn test; P<0.005) (not shown).
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Discussion |
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DNA damage can be caused directly, by exposure to UVR as a result of
absorbing photons of UVR, or indirectly, through the production of ROS
(Imlay and Linn, 1988;
Peak and Peak, 1990
). This DNA
damage can lead to the expression of p53
(Renzing et al., 1996
) and
apoptosis or cellular necrosis in many organisms. Our results show an increase
in the expression of SOD with exposure to UVR that is an indicator of an
increase in superoxide radicals and other forms of ROS
(Halliwell and Gutteridge,
1999
; Pourzand and Tyrell,
1999
). Oxidative stress is known to play a role in apoptosis
via several cell cycle genes such as p53
(Renzing et al., 1996
). Two
apoptotic pathways have been described and are known as the death-receptor
pathway and the mitochondrial pathway. The mitochondrial pathway is commonly
associated with DNA damage and upregulation or activation of the cell cycle
gene p53 (Hengartner,
2000
). Exposure to UVR also causes ROS production in the electron
transport chain of mitochondria (Gniadecki
et al., 2000
). Both the death receptor and mitochondrial pathways
converge at the mitochondria and the Bcl-2 family of genes where the release
of proapoptotic effectors (e.g. cytochrome c, ROS, caspase 9) occurs
and subsequently leads to the assembly of the apoptosome, which among other
things activates caspase-dependent DNase
(Green and Reed, 1998
;
Rich et al., 2000
).
The cell cycle checkpoint gene p53 allows a multicellular organism
to repair or delete cells exposed to agents that cause DNA damage, like
hypoxia, UVR, ROS or mutagens (Graeber et
al., 1996; Renzing et al.,
1996
; Clarke et al.,
1997
; Griffiths et al.,
1997
). Upregulation and expression of p53 allows DNA
editing and repair to occur followed either by normal cell division
(Polyak et al., 1997
) or
apoptosis (Hale et al., 1996
).
Cells with DNA damage caused by UVR and oxidative stress can survive but are
often retained in the G1/S phase of the cell cycle for long periods
of time (Geyer et al., 2000
).
These delays in cell division are the result of the expression of p53
and ultimately the downregulation of cyclin-dependent kinases
(Evan and Littlewood, 1998
).
An important pathway by which p53 facilitates an arrest in the cell
cycle is through the p21 protein. p21 is an inhibitor of a
wide range of kinases such as cdc2. p53 is also known to be
regulatory at the G2/M checkpoint through its effect on cyclin B1
after DNA damage (Innocente et al.,
1999
) and both p53 and p21 are required to
sustain a G2/M arrest after DNA damage
(Bunz et al., 1998
).
One diagnostic feature of apoptosis is extensive damage to chromatin and
DNA cleavage, leading to DNA fragments via an endogenous
endonuclease. The embryos of green sea urchins exposed to UVR exhibit both
direct effects of UVR on DNA, as observed through the accumulation of CPD
photoproducts, and the positive TUNEL assay, which indicates the occurrence of
DNA strand breaks caused by ROS (Imlay and
Linn, 1988; Pourzand and
Tyrell, 1999
). Subsequent to this DNA damage we observed a tight
coupling between p53 and p21 expression that is functionally
related to the delays in cell division observed in this species under similar
conditions (Adams and Shick,
1996
,
2001
;
Lesser and Barry, 2003
).
Additionally, the ultimate cell cycle regulator cdc2 shows a decrease in
concentration with increasing exposure to UVR in freshly fertilized embryos.
We believe this is either a consequence of the expression of p53 and
p21, or a direct consequence of exposure to UVR, or both. In fact,
inactivation of cdc2 has been shown to facilitate apoptosis induced by DNA
damage (Ongkeko et al., 1995
).
When the results of the TUNEL assay are examined it is apparent that some
cells, a significantly lower percentage, are apoptotic without having been
exposed to UVR. A functional apoptotic pathway has previously been shown in
sea urchin embryos and larvae (Voronina
and Wessel, 2001
; Roccheri et
al., 2002
) and the results reported here for green sea urchin
embryos reveal a significant increase in the number of apoptotic cells after
exposure to UVR. While the work of Voronina and Wessel
(2001
) suggests that the TUNEL
assay may underestimate the number of apoptotic cells, we feel that this
underestimation would be systematic throughout our experiments and thus not
affect the interpretation of UVR-induced apoptosis in the treatments described
above. The TUNEL assay results are also strongly supported by the differential
expression patterns of cell cycle and antioxidant proteins. Additionally, in
these experiments and other studies (Adams
and Shick, 2001
; Lesser and
Barry, 2003
) the abnormal morphologies observed (e.g. `packed'
blastula, blebbing, exogastrulation) are consistent with descriptions of
apoptotic morphology.
In summary, exposure of developing embryos of the green sea urchin to UVR causes a cascade of cellular events from DNA damage to apoptosis. Between these two events, well-described checkpoints and controllers of the cell cycle are regulated in a pattern that has been described for many metazoan systems. Additionally, the differential effects of UV-B versus UV-A are quite clear. Exposure to the shorter UV-B wavelengths, those affected by stratospheric ozone depletion, result in more DNA damage, higher levels of oxidative stress and greater expression of cell cycle genes that lead to apoptosis. We also see, however, significant effects on these processes from exposure to the UV-A portion of the spectrum, which has more total energy available and penetrates to deeper depths even in coastal temperate waters. Our results do not rule out the possibility of direct effects of UVR on critical proteins (e.g. cdc2), but the results presented here show that delays in cell division, abnormal development and, ultimately, the death of developing embryos begins with direct and indirect damage to DNA.
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Acknowledgments |
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