Mutagenesis vol. 26 no. 6 pp. 783–793, 2011
Advance Access Publication 8 August 2011
doi:10.1093/mutage/ger048
Induction and repair of DNA strand breaks and oxidised bases in somatic and
spermatogenic cells from the earthworm Eisenia fetida after exposure to ionising
radiation
Turid Hertel-Aas*, Deborah Helen Oughton,
Alicja Jaworska1 and Gunnar Brunborg2
*
To whom correspondence should be addressed. Department of Plant and
Environmental Sciences, Norwegian University of Life Sciences, PO BOX
5003, 1432 Aas, Norway. Tel: þ47 64 96 55 05; Fax: þ47 64 96 60 07; Email:
[email protected]
Received on March 2, 2011; revised on June 15, 2011;
accepted on July 2, 2011
Methods for analysing oxidised DNA lesions [formamidopyrimidine glycosylase (Fpg)-sensitive sites] in coelomocytes and
spermatogenic cells from the earthworm Eisenia fetida using
the Fpg-modified comet assay were established. The DNA
integrity (SSBs 5 strand breaks plus alkali labile sites and
Fpg-sensitive sites) in cells from E. fetida continuously
exposed to 60Co gamma-radiation (dose rates 0.18–43 mGy/h)
during two subsequent generations (F0 and F1) were
measured and related to effects on reproduction end points
which have already been reported. The data suggest a slight
increase of Fpg-sensitive sites in spermatogenic cells from
worms exposed at 11 mGy/h in the F0 generation but not in
F1, whereas reduced reproduction had been observed at dose
rates at or >4 mGy/h in F0 and at 11 mGy/h in F1. Using
acute X-rays (41.9 Gy/h), dose–response relationships were
established for SSBs in coelomocytes and spermatogenic cells
exposed in vitro. In vivo DNA repair was studied by
measuring the decrease in damage (SSBs and Fpg-sensitive
sites) in coelomocytes and spermatogenic cells isolated
from worms at different times (0–6 h) after acute
X-ray exposure (4 Gy). SSBs were repaired in coelomocytes
following biphasic kinetics, i.e. with a fast and a slow half-life
(t1/2) of 36 min (95%) and 6.7 h (5%), respectively. Fpgsensitive sites were repaired at considerably lower rates
(t1/2 5 4–5 h). In spermatogenic cells, SSB repair during the
first hour was observed but a half-life could not be estimated.
Repair of Fpg-sensitive sites could not be determined. In
general, a reduced repair of Fpg-sensitive sites suggests
a higher potential for accumulation of oxidised lesions,
compared to SSBs, in earthworms exposed to radiation and
other environmental contaminants. This is the first study
comparing DNA damage with reproduction in earthworms
exposed to ionising radiation.
Introduction
Wildlife can be exposed to ionising radiation both from natural
and anthropogenic sources. In the majority of cases associated
with routine releases of radionuclides, the exposure is chronic
Ó The Author 2011. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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783
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Department of Plant and Environmental Sciences, Norwegian University of
Life Sciences, PO BOX 5003, 1432 Aas, Norway, 1Department of Emergency
Preparedness and Environmental Radioactivity, Norwegian Radiation
Protection Authority, PO BOX 55, 1332 Osteraas, Norway and 2Department of
Chemical Toxicology, Division of Environmental Medicine, Norwegian
Institute of Public Health, PO BOX 4404 Nydalen, 0403 Oslo, Norway.
and dose rates are generally low (100 lGy/h) (1), but after an
accidental release, the levels can be significantly elevated.
Radiological protection has historically focused on human
protection. It is now, however, widely accepted that ecological
risks need to be addressed, and The International Commission
on Radiation Protection (ICRP) has proposed a new framework
in which the concept of reference animals and plants (RAPs)
are used to assess the impact of radiation (2). Based on their
radioecological properties and their important role in the soil
ecosystem, the earthworm has been selected as one of the
RAPs. As part of the risk assessment, there is a need for
information about the effects of chronic low dose exposure on
environmentally relevant end points such as reproduction in
many wildlife groups (3,4). Such studies are very costly and
time consuming and standard test methods have not been
established. As for other environmental stressors, monitoring
would benefit from predictive biomarkers for reproduction
effects. However, to date, the use of molecular markers in
radioecology has not been the subject of careful validation, and
it has been proposed that research in the area should include
both end points that are relevant for population dynamics as
well as molecular markers (5).
The obvious molecular biomarkers for early effects of
ionising radiation are various types of DNA lesions. Ionising
radiation interacts with DNA either directly by deposition of
energy or indirectly through the generation of intermediate
reactive oxygen species creating a spectrum of lesions. The
initial levels of the lesions in different cells may vary, for
example, in relation to the presence of low-molecular-weight
scavengers, intracellular oxygen tension (reviewed in ref. 6)
and physical protection due to chromatin packaging as with
spermatozoa (7,8). Among the lesions induced, double-strand
breaks (DSBs) are rare but considered to be of high
significance since one to two unrepaired DSBs are lethal while
they are mutagenic if misrepaired (9). In comparison with
DSBs, single-strand breaks are produced at much higher
frequencies but are generally repaired rapidly and mostly errorfree. Ionising radiation also produces frequent base lesions of
which oxidation of C8 in guanine is particularly important
since 8-oxo-7,8-dihydro-2#-deoxyguanosine (8-oxo-dG) is
potentially mutagenic leading to G:C to T:A transversion
mutations (reviewed in ref. 10). At least in some species and
tissues, 8-oxo-dG—which also arises due to normal oxidative
metabolism—is efficiently repaired via the base excision repair
(BER) pathway. If 8-oxo-dG is induced by ionising radiation, it
may be located within a clustered damage site (i.e. two or more
elemental lesions formed within 10–20 base pairs); the rate of
repair may then be reduced which in turn may increase the
mutagenic potential of the lesion (11,12). Clustered damage
sites may also lead to the formation of secondary DSBs during
repair ((13), reviewed in ref. 14).
Repair of DNA lesions whether induced by ionising
radiation or other agents varies between different tissues and
T. Hertel-Aas et al.
Materials and methods
Chronic exposure to c-radiation
E. fetida was continuously exposed during two successive generations (F0 and
F1) to external c-radiation from a 60Co source at dose rates from 0.18 to 43 mGy/
784
h as described in detail by Hertel-Aas et al. (27). The reproductive capacity of
adult F0 worms (20–23 weeks of age) was registered at specified time intervals
for 13 weeks. F1 offspring were irradiated from germ cell stage, and reproduction
was registered between 11 and 24 weeks of age. At the termination of exposure at
staggered intervals (1–3 days) for replicates, worms were transported from the
60
Co-irradiation facility in small boxes of soil kept on ice to the ‘comet lab’ (35
km away) where isolation of cells for the comet assay was started 1–2 h later.
Replicates were hence analysed on different days. If not stated otherwise,
coelomocytes and spermatogenic cells were isolated from the same worm to
investigate correlations between the levels of DNA damage in the two cell types;
such differences could have been masked by inter-individual differences. The
dose rates and numbers of F0 and F1 worms used are specified in Table I. For F0,
cells were isolated from worms exposed at the four highest dose rates (1.7–43
mG/h); spermatogenic cells from the 43 mGy/h samples were, however, not
available since the testicles and seminal vesicles were totally atrophied. At this
dose rate, the F0 worms stopped producing offspring after 4 weeks and no F1
individuals were available for analysis. Cells from the F1 4.2 mGy/h worms were
not included for comet analysis since they were exposed for an additional 8
weeks to examine the effects of prolonged treatment (27). Not all samples from
F0 were successfully analysed due to some comet gels, which could not be
scored (see Results and legend to Figure 1).
Acute exposure to X-rays
The worms used were sexually mature, 13–15 weeks of age and weighed 0.361
0.058 g obtained from the Norwegian Institute for Agricultural and
Environmental Research (Bioforsk) and cultivated as previously described (27).
Prior to exposure, worms were acclimatised in artificial Organisation for
Economic Co-operation and Development (OECD) soil (pH 6.3, 27% water
content) (28) for at least 4 days. For establishment of in vitro dose–response
relationships cells from one worm were used, in each of three independent
experiments. Cell aliquots were irradiated on ice with 0.5, 1, 2, 3, 6 or 10 Gy Xrays (260 KV, 0.5 mm Cu-filtering, dose rate: 41.9 Gy/h as estimated with
Fricke’s solution), followed by comet analysis.
In the in vivo repair experiments, worms were transferred to a 3-cm Petri dish
containing a moist filter paper and placed on ice/water for 5 min followed
by irradiation on ice with 4 Gy X-rays (dose rate 41.9 Gy/h). For determination
of initial levels of DNA damage (0 h), worms were kept on ice after irradiation
until cell isolation, whereas the other worms were quickly transferred to 30 g
OECD soil at 20.5–22°C to allow repair for 0.5, 1, 3 or 6 h. Irradiation was
performed so that cells from all individuals were isolated and analysed for DNA
damage at the same time (including a non-irradiated control worm). Three and
five independent experiments were performed for coelomocytes and spermatogenic cells, respectively.
Isolation of coelomocytes
After chronic exposure to c-radiation, coelomocytes were harvested as
described by Eyambe et al. (29) with minor modifications. Individual worms
were rinsed in saline (0.85 g/100 ml deionised water), dried with a paper towel
and weighed. Their posterior part was massaged to expel gut contents followed
by incubation in 2 ml extrusion medium [98% saline, 2% ethanol, 2.5 mg/ml
Na2EDTA and 10 mg/ml guaiacol glycerol ether (Sigma–Aldrich, St Louis,
MO, USA); pH 7.3] for 1 min at room temperature (RT). Worms were rinsed in
saline, transferred to OECD soil and kept at 9°C until isolation of
spermatogenic cells. Extruded cells were transferred to 18 ml ice cold RPMI
1640 medium with 25 mM HEPES and L-glutamine (Lonza, Veriviers,
Belgium) (5cell medium) plus 10% foetal bovine serum (FBS; PAA
laboratories GmbH, Pasching, Austria) and centrifuged for 10 min at 300 g, 4°C. After one more washing/centrifugation, pelleted cells in 0.5 ml
supernatant were counted, diluted and centrifuged again and resuspended in cell
medium.
For rapid isolation of cells in the acute X-ray experiments, coelomocytes
were collected using electrical stimulation, minimising the time from irradiation
of worms to comet analysis. Slightly modified from (30), worms were placed in
2 ml cell medium, and 4.5 V DC was applied on the cuticle surface for 2 5
sec. Excreted coelomocytes were collected on ice, counted and diluted.
Isolation of cells from seminal vesicles and testicles
For isolation of spermatogenic cells following chronic c-irradiation, we used an
enzyme-based procedure ((24) and Bustos-Obregon, personal communication)
with modifications related to for example enzyme incubation temperature and
duration and trypsin concentration. In the final procedure, testicles and seminal
vesicles were dissected out and transferred to 30 ll cell medium w/pyruvate
(0.1 mg/ml) and DL-lactate (0.05%) (both from Sigma–Aldrich), the tissue was
minced with scissors, followed by incubation with collagenase (Type 2, final
concentration 100 U/ml; Worthington Biochemical Corporation, Lakewood,
NJ, USA) and hyaluronidase (Type I-S, H3506, final concentration 300 U/ml;
Sigma–Aldrich) for 30 min at 30°C with gentle shaking every 5 min. At 20
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species and rates of repair are lesion specific. As an example,
rodent somatic cells seem to repair oxidised lesions [formamidopyrimidine glycosylase (Fpg)-sensitive sites] more
rapidly than human cells (15). We recently reported efficient
repair of alkylated and other base lesions via BER in germ cells
from both rats and humans (16), whereas repair of oxidised purine
lesions was efficient in rodent but not in human male germ cells
((17) and A.K. Olsen and G. Brunborg, unpublished results). Data
for humans strongly support the involvement of oxidised DNA
damage in the pathological process of male infertility, and sperm
from infertile men contains higher levels of 8-oxo-dG than control
individuals (reviewed in ref. 18). Taken together, these variations
highlight the importance of understanding oxidised lesion
induction and repair in the reproductive system.
The alkaline comet assay measures DNA damage (SSBs 5
mainly single-strand breaks and alkali labile sites) in single cells.
In addition, oxidised purines (primarily 8-oxo-dG) can be
detected by including treatment of the agarose embedded
nucleoides with a bacterial DNA Fpg extract (19). This way of
detecting oxidised DNA damage is considered to be the most
reliable and sensitive method currently available (reviewed in ref.
20). Although the comet assay is widely used in ecotoxicology
(especially aquatic), the assay has not yet been established as
a robust tool for monitoring purposes; this is partly due to the
lack of links between comet data and other ecologically relevant
end points (reviewed in ref. 21). The alkaline comet assay has
previously been applied to detect DNA damage in coelomocytes
(immune cells) from earthworms exposed in the laboratory to
artificially spiked soils (e.g. heavy metals and pesticides) [e.g.
(22–24)] or soils collected from polluted sites (e.g. polycyclic
aromatic hydrocarbons, heavy metals, uranium) [e.g. (25,26)]. Two
studies compared genotoxic effects in coelomocytes with changes
in earthworm reproduction (23,25). The end points had about the
same sensitivity following exposure of Eisenia fetida to polluted
soil from a former coking plant (25). Glyphosate (GLY) but not
chlorpyrifos (CPF) resulted in reduced reproduction in E. fetida,
whereas a significant increase in DNA damage only was observed
after exposure to CPF (23). Bustos-Obregon and Goicochea (24)
reported reduced reproduction and increased DNA damage in
spermatogenic cells from E. fetida following exposure to parathion,
and to our knowledge, this is the only study in which the comet
assay has been applied to this cell type. Genotoxic damage in germ
cells can be more directly linked to reproductive effects, and
measuring DNA damage in spermatogenic cells could potentially
be a better candidate as a biomarker for reproduction toxicity.
This study is part of a project investigating the effects of
chronic 60Co c-irradiation on survival, growth and reproduction
end points in the earthworm E. fetida at environmentally
relevant dose rates and during two generations (27). We have
developed and validated methods for measuring SSBs and Fpgsensitive sites using the comet assay in coelomocytes and
spermatogenic cells, and we here present results from analysis of
the chronically exposed worms. The persistence of induced
lesions would be expected to depend largely on DNA repair.
Little is known about efficiencies of DNA repair in earthworms,
and we therefore compared DNA repair in coelomocytes and
spermatogenic cells, following acute exposure to X-rays.
DNA damage in earthworms
Table I. Total absorbed doses, numbers and weights of worms and characteristics of cells isolated from F0 and F1 worms continuously exposed to c-radiation for
13 and 24 weeks, respectively
Dose rate (mGy/h)
Controls
1.7
4.2
11
43
—
0.39 0.06
8
—
—
—
3.6 0.5
0.38 0.03
3
8.6 1.3
0.42 0.03
3
23 3
0.38 0.05
4
85 13
0.46 0.03
5
70 7
43
—
—
57 9
22
64 16
22
65 7
21
58 9
33
76 4
10 3
—
—
71 4
11 5
82 6
73
75 7
10 4
—
—
—
6
0.60 0.09
0.73 0.11
4
0.56 0.06
7.0 1.1
4
0.61 0.04
—
—
—
45 7
4
0.69 0.03
—
—
—
68 7
32
71 7
32
72 10
42
—
—
76 5
32
—
—
79 4
42
78 9
53
82 3
42
—
—
85 6
74
—
—
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F0 generation
Absorbed dose (Gy)
Weight (g)
Worms (n)
Coelomocytes
Viability (%)
Hedgehogs (%)
Spermatogenic cells
Viability (%)
Hedgehogs (%)
F1 generation
Absorbed dose (Gy)
Worms (n)
Weight (g)
Coelomocytes
Viability (%)
Hedgehogs (%)
Spermatogenic cells
Viability (%)
Hedgehogs (%)
0.18
Average values SD are shown.
SSBs (SACA)
1.5
B)
50
% tail DNA
40
Total damage (+Fpg)
1.2
30
0.9
20
0.6
10
0.3
0
0.0
1.5
Fpg-sensitive sites
SSBs (-Fpg)
40
1.2
30
0.9
20
0.6
10
0.3
0
C 0.1
C)
50
1
C 0.1
10
D)
SSBs (-Fpg)
1.0
Total damage (+Fpg)
40
% tail DNA
0
0.8
30
1
10
50
Fpg-sensitive sites
1.0
40
0.8
30
0.6
0.6
20
20
0.4
0.4
10
10
0.2
0
0.0
0.2
0
C 0.1
1
10
Dose rate (mGy/h)
Number of lesions per 106 bp
50
0
C 0.1
1
Number of lesions per 106 bp
A)
10
Dose rate (mGy/h)
Fig. 1. DNA damage in cells from adult F0 worms exposed to c-radiation for 13 weeks. Lesions were measured as SSBs (Fpg) or as total damage (SSBs plus Fpgsensitive sites; þFpg), in coelomocytes (A) and spermatogenic cells (C) and are given as % tail DNA and number of lesions per 106 bp (left and right axis,
respectively). For coelomocytes, SSBs were also measured using the SACA. Each point represents the averaged medians of three technical replicates from one
worm, and the curves are through group means. Fpg-sensitive sites (þFpg minus Fpg values) in coelomocytes (B) and spermatogenic cells (D) are derived from
A and C, respectively. Coelomocytes (Fpg): control, n 5 7; 11 mGy/h, n 5 2; 43 mGy/h, n 5 5. Coelomocytes (SACA): control, n 5 8; 1.7 and 4.2 mGy/h,
n 5 2; 11 mGy/h, n 5 3; 43 mGy/h, n 5 5. Spermatogenic cells (Fpg): Control, n 5 8 worms; 1.8 mGy/h, n 5 3; 4.2 mGy/h, n 5 2; 11 mGy/h, n 5 4.
min, trypsin (T4665, final concentration 2325 U/ml; Sigma–Aldrich) was
added. After 10 min, samples were mixed with cold cell medium plus 10% FBS
1:1 on ice, followed by centrifugation at 900 g for 7 min and washing/
centrifugation twice (900 g 7 min) with cell medium plus 10% FBS. Finally,
cells were resuspended in cell medium and filtered through a 120-lm nylon
mesh. Samples were kept ice-cold if not stated otherwise. A fraction of each
suspension was fixed in 0.2% paraformaldehyde, for flow cytometric analysis
(Argus 100; Skatron Instruments AS, Lier, Norway). Fixed cells were stained
with Hoechst 33258, and the distribution of cells in different stages of the
spermatogenesis was estimated based on DNA content and light scatter. The
785
T. Hertel-Aas et al.
population, designated spermatogenic cells, contained 7% 2C cells; some of these
were somatic cells from surrounding tissue of the reproductive organs as verified
with anti-vimentin staining of the cytoskeleton and fluorescence microscopy.
For rapid isolation of spermatogenic cells in the acute X-ray experiments,
a mechanical procedure was used (31). Seminal vesicles and testicles were
dissected out and washed in ice-cold Merchant’s buffer (0.14 M NaCl, 1.47
mM KH2PO4, 2.7 mM KCl, 8.1 mM Na2HPO4 and 10 mM Na2EDTA; pH
7.4). The tissue was minced in the same buffer using a plastic piston in a
4.5-mm cylinder with a stainless-steel screen (0.4 mm) attached at the end. The
suspension was filtered through two layers of gauze and a 100-lm nylon mesh,
followed by centrifugation at 300 g for 5 min at 4°C. The pellet containing
a mixture of nuclei and intact cells was resuspended in Merchant’s buffer.
Viability of the cells, except spermatogenic cells isolated mechanically, was
estimated with fluorescence microscopic examination of samples 5 min after
staining with propidium iodide (5 lg/ml) and Hoechst 33342 (10 lg/ml).
786
Results
Chronic exposure to c-radiation
DNA damage (% tail DNA) was analysed in coelomocytes and
spermatogenic cells, isolated from F0 worms exposed to
c-radiation for 13 weeks and from their F1 offspring exposed
until 24 weeks. During these exposures, no radiation-induced
lethality was observed, but there was a time-dependent
decrease in reproduction at the two highest dose rates (27).
The total absorbed doses at the time of sacrifice are shown
in Table I. There was no significant difference in the weight
of worms (P 0.082) or the viability of coelomocytes
(P 0.054) and spermatogenic cells (P 0.24) between any
of the groups in either F0 or F1 (Table I).
Flow cytometric analyses of spermatogenic cell suspensions
showed mainly haploid cells (42 8% and 41 9% round and
elongating spermatids, respectively) plus smaller fractions of
diploid (spermatogonia, secondary spermatocytes and somatic
cells) and tetraploid (primary spermatocytes) cells (7 2%
and 7 3%, respectively) and cells in the S phase (2C–4C)
(3 1%). There was no significant difference (P 0.18)
between control and irradiated worms or between the two
generations. Although the cell preparation procedure should
not be considered quantitative for the cell types, it is worth
noting that, at 11 mGy/h, there was a higher fraction of
tetraploid cells in one F0 and in two F1 worms. Without
elongating spermatids, the flow cytometric distribution was
71 7%, 12 3% and 12 4% for round spermatids, diploid
and tetraploid cells, respectively. This was in good accordance
with the ploidies of nucleoids as scored in the comet assay.
Levels of SSBs were measured using both the standard
(SACA) and the modified (Fpg) comet assay of each
coelomocyte cell sample from F0 and F1, and spermatogenic
cells from F1 (Figures 1A, 2A and C). More SSBs were found
with the modified (Fpg) assay compared with SACA. Such
differences are also found for mammalian cells and probably
reflect unspecific cleavage at the elevated incubation temperature
(G. Brunborg, unpublished results).
F0 generation
The mean background levels of SSBs and Fpg-sensitive sites
in control worms (Figure 1) were not significantly different in
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Comet assay
SSBs and oxidised bases (5Fpg-sensitive sites) were measured as described (32)
with minor modifications. Aliquots of single cell suspensions (106cells/ml)
were carefully mixed 1:10 with 0.75% low melting point agarose (NuSieve;
Cambrex, Rockland, ME, USA) in phosphate-buffered saline (without Ca and
Mg, 10 mM Na2EDTA) at 34–35°C; 75 ll triplicates were applied to GelBond
films (Cambrex) placed on a cold Aluminium plate, using a brass/Teflon mould
giving 15-mm circular gels. Films were immediately transferred to lysis buffer
(2.5 M NaCl, 10 mM Tris–base, 100 mM Na2EDTA, 1% Na-lauroylsarcocine,
10% dimethyl sulfoxide (DMSO) and 1% Triton X-100; pH 10) at 4°C overnight.
For the standard alkaline comet assay (SACA), unwinding in electrophoresis
solution (0.3 M NaOH and 1 mM Na2EDTA; pH 13.2) was for 40 min at 4°C
followed by electrophoresis (0.8 V/cm on platform) for 30 min at 9°C in fresh
solution. After neutralisation in 0.4 M Tris buffer (pH 7.5) for 2 5 min and
rinsing in water (1 min), gels were fixed in 96% ethanol for 2 h, dried and stored
until scoring. For analysis of Fpg-sensitive sites, two additional steps were
incorporated after lysis: (i) films were washed in buffer (40 mM HEPES, 0.1 M
KCl and 0.5 M Na2EDTA; pH 7.6) at 4°C for 1 h; (ii) parallel films were placed in
this buffer with bovine serum albumin (0.2 mg/ml), pre-warmed to 37°C, either
without or with an Fpg-enzyme extract (final concentration, 0.71 lg/ml; (33)) for
1 h. For scoring, DNA was stained with SYBR Gold (Invitrogen, Carlsbad, CA,
USA) in TE-buffer (10 mM Tris–HCl and 1 mM EDTA; pH 8) at 1:12 500
dilution for 20 min at RT followed by 1-min wash in distilled water. Seventy-five
comets per replicate (225 per worm) were scored using a fluorescence microscope
(Leica DMLB, 40 magnification) with a CCD camera and the Komet 4.0
software (Andor Technology, Belfast, UK). The percentage of DNA in the comettail (% tail DNA) was chosen as the measure of DNA damage. Cells were selected
randomly; ‘hedgehogs’ (cells with small or non-existent head and large diffuse
tails containing .90% of the DNA) were not excluded. For the spermatogenic
cells, elongated spermatids and spermatozoa, which are easily identified, were not
analysed (both have highly compacted chromatin and specific procedures are
needed for their analysis in the comet assay (8)). Based on the distribution of % tail
DNA against the total fluorescence of each comet (proportional to the DNA
content), the ploidy specific level of DNA damage could be estimated for the
spermatogenic cells. In the Fpg-modified comet assay, Fpg-sensitive sites were
calculated by subtracting the median % tail DNA values without Fpg from the
median values with Fpg for the same sample. Net induction of DNA damage was
calculated by subtracting the background values of control (unexposed) samples.
Comet assay of the acutely exposed samples was performed as above with
some modifications. Electrophoresis was carried out in a different chamber with
buffer circulation, 2 12.5 5 25 V car batteries as power supply (0.88 V/cm
on the platform), and a different microscope (20 magnification) and scoring
system (Comet Assay IV; Perceptive Instruments Ltd, Suffolk, UK). One
experiment was scored with both types of software for comparison and there
was a good correlation in % tail DNA (slope 5 0.97, R2 5 0.95).
The number of lesions per 106 base pair (bp) was calculated as described by
Johansson et al. (34) using the slope (% tail DNA per Gy) of cell and
experiment-specific linear calibration curves (0–6 Gy) and assuming that 1 Gy
(X- or c-irradiation) induces 0.31 strand breaks/109 Da of cellular DNA (35).
Samples of cells from control earthworms were exposed to acute Xirradiation and always included as positive controls for SSBs. To establish
a positive control for induction of Fpg-sensitive sites, control cells were mixed
with Ro 12-9786 (final concentration 3 lM in 0.25% DMSO; a gift from Dr
Elmar Gocke, Roche Ltd, Basel, Switzerland) and after 1 min, the cells were
exposed to visible cold light for various times (1.25, 2.5 or 5 min). After
exposure, the suspension was diluted (1:1) with cold cell medium plus 10%
FBS followed by centrifugation (300 g 10 min, 4°C) and resuspension in cell
medium (32). Lesions were induced linearly up to 2.5 min of light exposure
with a net median damage level (i.e. minus DMSO vehicle control) of 55% tail
DNA. As a routine positive control, light exposure for 1.25 min was used.
Statistical analysis
To evaluate whether there was any differences in weight of worms and viability
of cells from the different exposure groups, the data were analysed with
analysis of variance (ANOVA) in the General Linear model (GLM) mode of
Minitab software (MINITABÒRelease 14; Minitab Inc., State College, PA,
USA). There is currently no consensus on a standard statistical method for the
analysis of comet data (36). In the chronic exposure experiment and the acute
repair study, each individual worm was considered as the experimental unit.
Median % tail DNA values from each technical replicate were chosen since
medians are less sensitive to outliers than means (37). In all experiments, the
averaged medians from three technical replicates from each worm were
subjected to statistical analysis. The effects of chronic c-irradiation on DNA
integrity were evaluated by comparing all groups using the ANOVA GLM
followed by Tukey’s post hoc test. In addition, trend analysis in the form of
linear regression was performed (Minitab) and a significant dose–rate response
relationship was indicated by a slope significantly different (P , 0.05) from
zero. In cases when assumptions of normality of residuals and/or homogeneous
variance were violated (Anderson Darling test, P , 0.01 and Levene#s test, P
, 0.05, respectively), the data was log10-transformed.
To evaluate if there was any difference in the in vitro dose–response after acute
X-irradiation between coelomocytes and spermatogenic cells, the slopes of the
linear part of the polynomial regression curves from the three independent
experiments were compared using the t-test (Minitab). In the repair experiments,
one- or two-phase exponential regression lines were fitted to the data using
GraphPad Prism (GraphPad Prism 5, GraphPad Software Inc., La Jolla, CA, USA).
DNA damage in earthworms
coelomocytes and testicular cells (P 0.24), but the interindividual variation of Fpg-sensitive sites was much higher
for the former, resulting in a lower precision of measuring
these lesions in coelomocytes [coefficient of variance (CV) 5
100%] compared to spermatogenic cells (CV 5 40%).
After 13 weeks of adult F0 exposure, low but significant
increases were observed for some of the end points (Figure 1).
When significant dose–rate response trends were found using
linear regression, the goodness of the fit (R2 adj.) was generally
low (all regression parameters are shown in Supplementary
Table I, available at Mutagenesis Online). For coelomocytes,
SSBs and Fpg-sensitive sites were not significantly different
between the groups (P 0.077), but there was a weak dose–rate
response relationship for SSBs (slope 5 1.02, P 5 0.009, R2 adj.
5 28.5%). The total damage level (SSBs þ Fpg-sensitive sites)
was significantly higher in coelomocytes from worms exposed
at 43 mGy/h compared to controls (P 5 0.032) and 11 mGy/h
(P 5 0.029) and a dose–rate response relationship was indicated
(slope: 0.37, P 5 0.01, R2 adj. 5 38%). It should be noted,
however, that the Fpg coelomocyte samples were successfully
analysed only for controls and the two highest dose rates (with
n 5 2 for 11 mGy/h).
With spermatogenic cells (Figure 1C and D), there was
no effect of radiation (highest dose rate 11 mGy/h) on SSBs
(P 5 0.48 between groups, and 0.14 for regression). There
was, however, a significant increase in Fpg-sensitive sites at
11 mGy/h compared to the controls (P 5 0.024), and a dose–rate
response relationship for these lesions was found (slope: 0.74, P
5 0.005, R2 adj. 5 39%); the inter-individual variation at 11
mGy/h was relatively high (CV 5 61%). For spermatogenic
B)
SSBs (-Fpg)
% tail DNA
40
1.2
Total damage (+Fpg)
30
0.9
20
0.6
10
0.3
0
0.0
50
1.5
Fpg-sensitive sites
40
1.2
30
0.9
20
0.6
10
0.3
0
C 0.1
C)
50
1
% tail DNA
40
C 0.1
10
D)
SSBs (SACA)
1.0
SSBs (-Fpg)
0
Total damage (+Fpg)
0.8
30
1
10
50
Fpg-sensitive sites
1.0
40
0.8
30
0.6
0.6
20
20
0.4
0.4
10
10
0.2
0
0.0
0.2
0
C 0.1
1
10
Dose rate (mGy/h)
Number of lesions per 106 bp
1.5
SSBs (SACA)
Acute exposure to X-rays
DNA damage after in vitro exposure of cells. The viability of
coelomocytes prepared using electric simulation for in vitro
exposure was 84 3%. Figure 3 presents net DNA damage
0
C 0.1
1
Number of lesions per 106 bp
50
F1 generation
For spermatogenic cells from control worms, a significantly
lower level of SSBs was detected in F1 (Figure 2C) compared
to F0 (Figure 1C), whereas the number of Fpg-sensitive sites
(Figure 1D and 2D) was similar (P 5 0.005 and P 5 0.21,
respectively). Exposure to c-radiation for 24 weeks did not lead
to significant changes in the DNA integrity in coelomocytes
when groups were compared (P 0.126) although a weak
dose–response relationship was indicated for SSBs measured
with SACA (slope: 0.21, P 5 0.035, R2adj. 5 20%). For the
spermatogenic cells, no major radiation-induced differences
were found, although statistical analysis was complicated by
non-normality and heterogeneous variance (Supplementary
Table I is available at Mutagenesis Online).
The response of the two cell types could not be thoroughly
correlated in the same individual as intended, since (i) the male
reproductive organs were totally atrophied in F0 worms
exposed to 43 mGy/h and (ii) coelomocytes samples were
not successfully analysed for all dose rates (Figure 1). In F1,
lower damage levels were detected in spermatogenic cells; no
consistent correlation patterns between levels in the two cell
types were observed.
10
Dose rate (mGy/h)
Fig. 2. DNA damage in cells from adult F1 worms exposed to c-radiation for 24 weeks. Lesions were measured as SSBs, using either the standard alkaline (SACA)
or the modified (Fpg) comet assay, and as total damage, i.e. SSBs plus Fpg-sensitive sites (þFpg) in coelomocytes (A) and spermatogenic cells (C). Fpg-sensitive
sites (þFpg minus Fpg values) in coelomocytes (B) and spermatogenic cells (D) are derived from A and C, respectively. See legend to Figure 1 for further details.
Control: n 5 6; 0.18–11 mGy/h, n 5 4.
787
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A)
cells, the data set is somewhat more complete than for
coelomocytes (see legend to Figure 1).
T. Hertel-Aas et al.
measured in the cells after X-irradiation. For both cell types,
the dose-dependent increase in the induction of SSBs was
linear up to 6 Gy, and the slopes of the linear part of the
regressions curves were not significantly different (P 5 0.183).
The spermatogenic cell population comprised 68 6% 1C,
21 4% 2C and 11 4% .2C as analysed in the comet assay.
Ploidy specific dose–response curves did not indicate any
difference in the induction of SSBs between the cell types (data
not shown).
Fpg treatment gave only minor increases in the levels of
damage compared to SSBs; no clear dose–response was
observed for Fpg-sensitive sites in either coelomocytes or
spermatogenic cells (Figure 3). This is in accordance with other
observations that Fpg is unable to detect and quantitatively
cleave oxidised base lesions in the presence of large numbers
of SSBs and/or clustered lesions (discussed in refs 38,39).
SSBs (-Fpg)
80
Total damage (+Fpg)
2.0
1.6
y = −0.80 x 2 + 15.0 x + 0.86
R = 0.96
% tail DNA
2
60
1.2
40
0.8
y = −0.68 x + 14.4 x − 1.19
2
20
0.4
R 2 = 0.98
0
0.0
0
B)
100
2
4
6
SSBs (-Fpg)
8
10
Total damage (+Fpg)
1.6
y = −1.16 x 2 + 19.6 x + 1.78
80
% tail DNA
Number of lesions per 106 bp
100
R 2 = 0.98
1.2
60
0.8
40
y = −0.89 x 2 + 17.2 x − 0.85
20
0.4
R 2 = 0.98
0
Number of lesions per 106 bp
A)
0.0
0
2
4
6
Dose (Gy)
8
10
Fig. 3. Dose–response relationship for DNA damage after acute X-ray
irradiation of coelomocytes (A) and spermatogenic cells (B). Cells were
derived from one worm, and aliquots were irradiated on ice followed by comet
analysis. Lesions were measured as SSBs (Fpg) or as total damage, i.e. SSBs
plus Fpg-sensitive sites (þFpg). Each point represents the averaged medians of
three technical replicates in one independent experiment (n 5 3). Background
levels of SSBs (8 and 5% tail DNA) and total damage levels (15 and 9% tail
DNA) in coelomocytes and spermatogenic cells, respectively, have been
subtracted. Polynomial regression curves for SSBs (stipulated line) and total
damage levels (solid line) are shown.
788
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Induction and repair of DNA damage after in vivo exposure.
Worms were irradiated with 4 Gy on ice followed by
incubation at RT. This approach was used since cells in their
natural environment would be expected to have optimal
conditions for repair. In the subsequent isolation of cells for
comet analysis (which lasted for 67 or 85 min until lysis, for
coelomocytes and spermatogenic cells, respectively), cells
were kept ice-cold at all steps to prevent repair. In these
experiments, the viability of coelomocytes was 90 5% with
no significant difference between controls and irradiated worms
(P 0.81).
Cell suspensions from control worms were irradiated with 4
Gy and the level of DNA damage was compared with the initial
(t 5 0 h) value as measured in cells from worms which were
exposed to the same dose. The number of SSBs in the in vivo
samples were 20% lower for both cell types compared to in
vitro, suggesting that some repair did occur during sample
processing after irradiation. Frequency distributions of comets
in groups with specified levels of DNA damage show clear
differences between coelomocytes and spermatogenic cells at
various times of repair (Figure 4).
For coelomocytes, curves containing one or two exponential
terms could be fitted to the experimental data (R2 5 0.90 for
both models) describing the decline in SSBs with time. The
half-life (t1/2) of SSBs using monophasic repair kinetics was 40
min. For the biphasic model (Figure 5A), 95% of the initial
damage underwent rapid repair with t1/2 5 36 min, whereas t1/2
for the slow component was 6.7 h. The uncertainties associated
with the calculated parameters were, however, considerable
larger for the latter model (Supplementary Table II is available
at Mutagenesis Online).
At time zero after in vivo irradiation—unlike in vitro
irradiation—the Fpg treatment gave 44% more DNA damage
compared to SSBs alone (Figure 5A). The Fpg-sensitive sites
increased slightly after 0.5-h repair incubation (Figure 5B); an
increase at short repair times has also been observed for other
cell types (15,39), probably reflecting that when a fraction of
SSBs has been repaired, the Fpg enzyme is able to detect and
cleave the base lesions more quantitatively (39). Curve fitting of
the time-dependent data for Fpg-sensitive sites was attempted
from time point 0.5 h (maximum level) using various regression
models. Linear one- or two-phase exponential decay gave similar
half-lives but limited fit (R2 5 0.50–0.55). For the exponential
models, t1/2 was 3.7 h (one-phase) or 5.0 h (major fraction
(77%), two-phase); again there are considerable uncertainties
(Supplementary Table II available at Mutagenesis Online).
Figure 5C shows time-dependent repair of DNA damage in
spermatogenic cells. The samples from different worms showed
a relatively high variability in DNA damage initially and during
repair. Within each of the five experiments, there was no clear
time-dependent decrease in SSBs, and the damage levels did not
consistently reach the control levels even after 6 h. A precise halflife could not be estimated by regression analysis using different
models. Figure 4B suggests that some cells completely repaired
the radiation-induced SSBs within the first hour after irradiation,
but afterwards, there was almost no redistribution of cells
between the major damage groups.
High inter-individual variation in the measured damage
levels could be related to different repair capacities among
spermatogenic cells of different ploidy combined with variation
in the distributions of such cell types between independently
isolated cell suspensions. Analysis of ploidy-specific comet
data did, however, not reveal consistent cell type-specific
differences in DNA repair, and no consistent reduction in the
CVs for 1C, 2C and .2C ploidy-specific comet data was
found, when comparing with CV for the mixed cell population
(data not shown).
For the Fpg-sensitive sites (Figure 5D), a further analysis of
the data gave no clear indication of repair.
DNA damage in earthworms
% of comets
% of comets
% of comets
100
SSBs (- Fpg)
Total damage (+ Fpg)
B)
100
Control
50
SSBs (- Fpg)
Total damage (+ Fpg)
Control
50
0
0
100
100
t = 0h
50
t = 0h
50
0
0
100
100
t = 0.5h
50
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% of comets
% of comets
% of comets
A)
t = 0.5h
50
0
0
100
100
t = 1h
50
t = 1h
50
0
0
100
100
t = 3h
50
t = 3h
50
0
0
100
100
t = 6h
50
t = 6h
50
0
0
10 20 30 40 50 60 70 80 90 100
10 20 30 40 50 60 70 80 90 100
% tail DNA
% tail DNA
Fig. 4. Frequency distribution of comets from coelomocytes (A) and spermatogenic cells (B) in groups with different levels of DNA damage (% tail DNA). Worms
were irradiated on ice with 4 Gy X-rays and incubated at RT for 0.5–6 h to allow repair. Cells from all worms (including 0 h and controls) were isolated and
subjected to comet analysis. Lesions were measured as SSBs (Fpg) or as SSBs plus Fpg-sensitive sites (þFpg). Means SD of independent experiments are
shown. Coelomocytes (n 5 3), spermatogenic cells (n 5 5, except at t 5 0 and 3 h for which n 5 4); 225 cells were scored per worm.
Discussion
There are few studies using the comet assay to monitor cellular
DNA damage in non-human species environmentally exposed
to ionising radiation, and none directly linking the measurement of DNA integrity with reproduction end points (26,40). In
the present study, we used this assay to measure the DNA
integrity of somatic and spermatogenic cells, from earthworms
exposed to chronic or acute radiation. DNA lesions were
compared to reproduction end points in chronically exposed
worms, and Fpg-sensitive sites (oxidised purines) were
measured for the first time in E. fetida. The background levels
of Fpg-sensitive sites ranged from 0.13 to 0.33 and from 0.05
789
SSBs (-Fpg)
70
1.4
% tail DNA
Fpg-sensitive sites
70
1.4
1.2
60
1.2
1.0
50
1.0
0.8
40
0.8
30
0.6
30
0.6
20
0.4
20
0.4
10
0.2
10
0.2
0.0
0
60
50
y = 28e ( −1.16 x ) + 1.6e ( −0.10 x )
40
0
0
C)
B)
Total damage (+Fpg)
1
2
3
4
5
SSBs (-Fpg)
70
0.0
0
6
1.6
D)
1
2
3
4
5
6
Fpg-sensitive sites
70
1.6
Total damage (+Fpg)
% tail DNA
1.2
50
1.0
40
1.4
60
1.2
50
1.0
40
0.8
30
0.6
20
0.4
10
0
0
1
2
3
4
5
0.8
30
0.2
10
0.0
0
6
Time after irradiation (h)
0.6
20
0.4
0.2
0.0
0
1
2
3
4
5
6
Time after irradiation (h)
Fig. 5. Induction and repair of DNA damage in coelomocytes (upper panels) and spermatogenic cells (lower panels) from worms exposed to acute X-rays (4 Gy).
Worms and cells are the same as described in legend to Figure 4. Lesions were measured as SSBs (Fpg) and SSBs plus Fpg-sensitive sites (þFpg). Net Fpgsensitive sites (þFpg minus Fpg values) in coelomocytes (B) and spermatogenic cells (D) are derived from A and C, respectively. Each point represents the
averaged median of three technical replicates from one worm in one experiment after subtraction of background levels (mean values: Fpg, 1.5 and 3.5; þFpg, 6.4
and 12% tail DNA for coelomocytes and spermatogenic cells, respectively). The two-phase exponential regression curve (solid) for SSBs is shown in (A); all other
lines represent means of the damage levels. Coelomocytes: n 5 3; spermatogenic cells: n 5 5, except at t 5 0 and 3 h for which n 5 4. (At t 5 0, one untypically
low value was removed from data analysis; % tail DNA514, versus 29–66 in the 4 other experiments (1.7 standardised SD).
to 0.13 lesions/106 bp (Figures 1–3 and 5) in control
coelomocytes and spermatogenic cells, respectively. These
levels are similar to what has been reported for human blood
cells (0.68/106 bp) (41) and rat and human testicular cells (0.14
and 0.3–0.4/106 bp, respectively (17). A substantial increase
above these levels is tolerated since we observed higher levels
of Fpg-sensitive sites in the testis and liver of Ogg1/ mice,
which are fertile and apparently healthy ((42), and A.K. Olsen
and G. Brunborg, unpublished results). However, in these
studies, the level of oxidised purines in the fertilising sperm is
not known.
Exposure to chronic c-radiation
DNA SSBs induced by chronic exposure would be in dynamic
equilibrium with their removal, and since their repair is rapid in
most cell types, the steady-state level is expected to be very
low at moderate dose rates. Correspondingly, we found no or
very low increase in the level of SSBs by chronic irradiation in
coelomocytes and spermatogenic cells (Figures 1A and C and
2A and C) which is also in accordance with the rapid repair in
coelomocytes after acute X-ray exposure (Figure 5A). At the
highest dose rate (43 mGy/h), the total level of DNA damage,
i.e. SSBs plus Fpg-sensitive sites, was increased in coelomocytes from the F0 worms. These worms had stopped producing
viable cocoons after 4 weeks of exposure and their testicles and
seminal vesicles were totally atrophied at the time of cell
isolation (27). A slight increase was also observed of oxidised
790
lesions in spermatogenic cells from F0 at 11 mGy/h. This was
the lowest dose rate causing a pronounced and significant
reduction in the hatchability (to 25%) of cocoons produced
during the last 9–13 weeks of exposure (27) although there was
an indication of reduced reproduction capacity also at 4.2 mGy/h.
For the F0 generation, the comet-analysis should, however, be
interpreted with some care—particularly for coelomocytes—
since the data set is limited and does not represent all the dose
rates employed.
In F1 worms, the reproduction capacity was severely
reduced at 11 mGy/h during the whole registration period;
the total dose absorbed by these worms was twice as high as in F0
(Table I). This exposure was, however, not accompanied by any
apparent increase in oxidised DNA lesions in the spermatogenic
cells. The data therefore do not suggest a consistent correlation
between the reduced reproduction at 11 mGy/h and the level of
oxidised DNA lesions. In general, oxidised lesions may be
removed from the cell population not only by DNA repair but
also via apoptosis, cell turnover and dilution via cell
replication. The apparent different responses in F0 and F1
could reflect variations in such processes, which may be related
to age and/or exposure history.
Induction and repair of DNA damage after acute exposure to
X-rays
Induction of lesions and their post-irradiation removal were
studied after exposure to high dose rate X-rays. The rationale
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1.4
60
Number of lesions per 106 bp
A)
Number of lesions per 106 bp
T. Hertel-Aas et al.
DNA damage in earthworms
the sperm [e.g. (43)]. In human and rodent spermatocytes and
round spermatids, but not in mature elongated spermatids, SSBs
are repaired at the same rates (t1/2–10 min) as in several somatic
cell types (43). Our study indicates repair of SSBs in crude
spermatogenic cells during the first hour following acute X-ray
exposure, but at later times, the average damage level seems to
plateau (Figure 5C). High inter-individual variability was
observed and no half-life could be estimated. Ploidy-specific
comet data did not reveal significant differences in repair
capacity among the different stages. The data gave no clear
indication of repair of Fpg-sensitive sites in spermatogenic cells
after acute exposure to X-rays (Figure 5D). As discussed above,
there is probably a varying ability to repair oxidised lesions in
spermatogenic cells between species (17), and a possible lack of
such repair in the earthworm should therefore be considered;
there is, however, no data available on activities or expression of
DNA repair proteins in germ or somatic cells in these species.
On the other hand, the low levels of Fpg-sensitive sites in
spermatogenic cells arising spontaneously and after chronic
irradiation would suggest considerable repair and/or apoptosis.
In general, the usefulness of the modified comet assay to
detect DNA damage (SSBs and oxidised lesions) as environmental biomarkers will depend on various factors such as the
background levels and the inter-individual variability in control
animals, the sensitivity of the end point to exposure, and—
most importantly—correlation with ecologically relevant end
points. The comet assay as used in the present study seems to
be less sensitive compared to reproduction end points since we
were able to demonstrate reduced reproduction at dose rates
which did not lead to detectable increases in DNA damage. The
lowest dose rate used (0.18 mGy/h, F1) is within the same
order of magnitude as dose rates estimated for soil invertebrates
at sites heavily contaminated by uranium mining (51) or
radioactive waste (52). The lowest dose rate inducing severe
effects on reproduction (11 mGy/h) and significant increase in
Fpg-sensitive sites in spermatogenic cells in F0 is high
compared to most environmental situations but might be
encountered after serious accidents such as Chernobyl (53).
The usefulness of the comet assay for earthworms in
biomonitoring should not, however, be evaluated based on
effects of ionising radiation alone. The lesions measured by the
standard and Fpg-modified comet assay are not specific for
ionising radiation and can be induced by a range of
environmental contaminants. Significant increases in DNA
damage (SSBs) in coelomocytes, as measured with the alkaline
comet assay, have been reported for Eisenia andrei after
exposure (1–56 days) to soil from an uranium mine highly
contaminated with heavy metals and radionuclides (e.g. 238U,
234
U, 230Th, 226Ra) (26). Some metals are known to inhibit
DNA repair enzymes (54), which also highlight possible
synergy between effects of radiation and chemical contaminants. Further evaluations should involve combined exposures,
whereas the quick cell isolation methods described (i.e.
electrical stimulation and a refined mechanical procedure)
could potentially increase assay sensitivity. The present results
underline the need for investigation of the time-dependent
change in the DNA damage response after increasing times of
exposure, both within and between generations since the steady
state levels of damage might change with time as protective
mechanisms might be induced.
In conclusion, the exposure set-up which we used for
chronic irradiation seems very useful for studying the effects of
environmental exposures. Combined with the comet assay, it
791
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of these experiments was to provide a background for
understanding the effects of chronic exposure and also to
allow comparison of DNA repair in other species and cell
types. Protocols involving rapid isolation of coelomocytes and
male germ cells from E. fetida were used. The results suggested
no significant difference in the in vitro induction of SSBs after
acute X-ray irradiation in coelomocytes compared to spermatogenic cells or between spermatogenic cells of different
ploidy. This is in accordance with the observations for
spermatocytes, round spermatids and bone marrow cells from
hamster (43).
Oxidised DNA lesions in the form of Fpg-sensitive sites are
induced by ionising radiation at approximately the same
frequencies as single-strand breaks, although the former lesions
are not detected quantitatively directly after acute X-irradiation
(Figure 3) as discussed in the Results section. During chronic
irradiation SSBs are continuously repaired and it is therefore
likely that Fpg-sensitive sites can be quantitatively detected in
such exposure situations.
In general, there is little information of DNA repair
including their time kinetics in wildlife species (discussed by
(21)). To our knowledge, this is the first study of repair kinetics
of SSBs and Fpg-sensitive sites in earthworms. Even with
careful control of the worms’ temperature, some of the initial
SSBs measured right after irradiation (t 5 0 h) were apparently
repaired during cell preparation suggesting that the reported
half-lives (Figure 5A) might be longer than in reality. The
underlying basis for a fast (t½ 5 36 min) and a slow (t½ 5 6.7
h) repair kinetics suggested for coelomocytes may be related to
the localisation of the damage (in relaxed versus condensed
chromatin) (44), or to various types of damage being repaired
at different rates (separated single SSBs versus DSBs and/or
clustered damage sites) [(11) discussed by (45)]. Similar twocomponent repair kinetics of radiation-induced SSBs have been
observed in cells from a number of other species such as yeast
(46), Drosophila melanogaster (47), rodents and humans (45).
However, the in vivo rates of repair in E. fetida coelomocytes
seem to be lower than in cells from these species [e.g. for
D.melanogaster, t½ fast/slow: 5 min/20 min (47); human
peripheral blood mononuclear cells, t½ fast: 6–12 min (45)].
The repair half-life was estimated as 20–30 min by Brash and
Hart (48) in rats in vivo, at a high dose (50 Gy) of X-rays using
alkaline sucrose gradients. In plants, slow in vivo repair of
SSBs was reported for tobacco seedlings and wood small-reed
(Calamagrostis epigejos), with a half-life of 52 and 101 min,
respectively (49,50).
Figure 5B suggests that Fpg-sensitive sites are repaired in
coelomocytes, but this occurs at considerably lower rates than
for SSBs, which is in accordance with observations for humans
and rodents (15,39). Repair of radiation-induced (4 Gy X-rays)
Fpg-sensitive sites was faster (t1/2 5 25 10 min) in various
cultivated rodent cells compared to human cells (t1/2 5 80 20
min) (15), but both species seem to repair these lesions more
rapidly than E. fetida (t1/2 5 4–5 h). An accurate estimation of
rates of repair of Fpg-sensitive sites is, however, complicated
by the inefficient detection of oxidised lesions at time zero, and
considerable uncertainties are associated with the given halflife. Biphasic repair kinetics were observed for the rodent and
human cell lines (15), whereas in the present study, limited fits
were achieved using exponential mono- or biphasic models.
It is known that the ability to repair certain DNA lesions
decreases as the spermatogenesis proceeds with DNA chromatin
becoming highly compacted together with reduced cytoplasm in
T. Hertel-Aas et al.
allowed us to investigate possible accumulation of oxidised
DNA lesions that are likely to accumulate in nature, in
particular in species with low capacity for repair of such
lesions. This is the first report of radiation-induced oxidised
lesions in somatic and spermatogenic cells of earthworms. The
persistence of DNA lesions in the worms could be directly
compared with their reproductive capacities, which were
described in our previous report (27).
Supplementary data
Supplementary Tables I and II are available at Mutagenesis
Online.
Funding
Acknowledgements
We thank Richard Wiger for characterisation of the spermatogenic cell
suspensions by flow cytometry and anti-vimentin staining, and Dr Elmar Gocke
(Roche, Switzerland) for supplying the Ro 12-9786 compound.
Conflict of interest statement: None declared.
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