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GENOTOXICITY OF MARINE SEDIMENTS IN THE FISH HEPATOMA CELL LINE PLHC-1 AS
ASSESSED BY THE COMET ASSAY
Maja Šruta*, Luka Travenb,c, Anamaria Štambuka, Sonja Kralja, Roko Žajad, Vladimir Mićovićb,c, Göran
I.V. Klobučara
a
Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb,
Croatia
b
Department of Environmental Medicine, Medical Faculty, University of Rijeka, Braće Branchetta
20a, 51000 Rijeka, Croatia
c
Teaching Institute of Public Health of the Primorsko-goranska county, Krešimirova 52a, 51000
Rijeka, Croatia
d
Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Ruđer
Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia
*
corresponding author:
Maja Šrut
Department of Zoology
Faculty of Science
University of Zagreb
Rooseveltov trg 6
10000 Zagreb
Croatia
Phone: +385 1 4877700, Fax: +385 1 4826260
e-mail: msrut@zg.biol.pmf.hr
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ABSTRACT
The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma
fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine
sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and
ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent
dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model
chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest
observed genotoxic potential was additionally extracted using various solvents in order to test which
class of compounds, according to their polarity, is mostly responsible for the observed genotoxic
effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but
the highest level of DNA damage was determined after exposure to sediment extract obtained with the
solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results
indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and
encourage the use of fish cell lines as versatile tools in ecogenotoxicology.
Keywords: Comet assay, PLHC-1 cell line, marine sediments, DNA damage, extraction solvent
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1. INTRODUCTION
Sediments are the main sink for anthropogenic contaminants in aquatic environments. Toxic
compounds can be continually re-introduced to the water column via re-suspension and trophic
transfer causing adverse effects to freshwater and marine biota and thus represent a long-term source
of pollution (Chen and White, 2004). Therefore, in order to undertake evidence-based preventive and
remedial actions it is of paramount importance to assess and characterize the ecological risks posed
by contaminated aquatic sediments.
When considering the influence of toxicants on different taxonomic groups, fish can be
considered as one of the key groups which should be evaluated in terms of ecological risk. Fish
represent the most diverse group of vertebrates which occupy various ecological niches in aquatic
ecosystems and play a crucial role in the transfer of energy between different trophic levels. Thus,
understanding the actions of toxicants on fish can provide key insights for evaluating the overall health
of the aquatic environment (Bols et al., 2005).
The genotoxic potential of sediments has been assessed either using whole sediments or
sediment extracts. Whole sediments are usually employed when studying the effects on whole
organisms and such approach has been applied to fish both in situ (Kammann et al., 2000; Barbee et
al., 2008) and ex situ (Kilemade et al., 2004; Inzunza et al., 2006; Costa et al., 2008). On the other
hand, sediment extracts are mostly implemented on fish cell lines which have been widely used in the
characterization of ecological risk since they offer several advantages compared to intact animals.
Although the employment of whole organisms has greater ecological importance, the in vitro
experiments can be conducted in a well-controlled and predetermined environment, the results are
obtained more rapidly, with less costs and are more reproducible (Bols et al., 2005).
The Comet assay is a well-known and widely used method for assessment of genotoxicity. It
has a broad applicability to aquatic animals and can be used for both the in vitro and, in vivo
exposures, including the in situ surveys (Lee and Steinert, 2003). Despite its wide applicability, a lack
of standardization is still an important issue that has to be dealt with in order to allow for a direct
comparison of data obtained from different research groups (Ejchart and Sadlej-Sosnowska, 2003;
Seitz et al., 2008). So far the Comet assay has been successfully applied on a number of different fish
cell lines (Nehls and Segner, 2001; Avishai et al., 2002; Kamer and Rinkevich, 2002; Kammann et al.,
2004; Klee et al., 2004; Vevers and Jha, 2008) but it has been scarcely used in assessing sediment
genotoxicity. Kammann and colleagues used the Comet assay on the EPC (epithelioma papulosum
cyprini) fish cell line derived from a skin tumor of carp (Cyprinus carpio) to characterize the
genotoxicity of sediment extracts from the Baltic and North Sea (Kammann et al., 2001; Kammann et
al., 2004). For the assessment of genotoxicity of freshwater sediments the RTL-W1 permanent cell line
derived from rainbow trout liver (Oncorhynchus mykiss) has been used (Kosmehl et al., 2004; Keiter et
al. 2006; Seitz et al., 2008; Rocha et al., 2009).
The permanent fish cell line PLHC-1 derived from a hepatocellular carcinoma of the
topminnow Poeciliopsis lucida is one of the commonly used cell lines for toxicity screening of
chemicals and environmental samples. This cell line posses xenobiotic-metabolizing capacity and
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contain aryl hydrocarbon (Ah) receptor which allows for testing of indirect carcinogens as well (Hahn et
al., 1993). Different toxicological endpoints have been evaluated in these cells including cytotoxicity
(Babich et al., 1991), cell growth (Brüschweiler et al., 1995; Fent and Bätscher, 2000) and xenobiotic
metabolism (Hahn et al., 1993; Caminada et al., 2008; Thibaut et al., 2009). The induction of CYP1A
in PLHC-1 cells has been used to characterize the ecotoxicological risk posed by contaminated water
and marine and freshwater sediments (Huuskonen et al., 1998a, 1998b, 2000; Traven et al., 2008).
However, to our knowledge genotoxicity has not yet been determined in the PLHC-1 cell line upon the
exposure to either toxic agents or environmental samples.
The aim of this study was to establish the use of the Comet assay on the PLHC-1 cell line as
an ecotoxicological tool which can be used for screening contaminated marine sediments for the
presence of genotoxic compounds. We have tested the system using both model chemicals
(benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and environmental samples. Furthermore
we tested sediment extracts obtained using various solvents (dichloromethane (DCM), methanol,
cyclohexane, DCM/methanol) in order to test which class of compounds, according to their polarity, is
mostly responsible for the observed genotoxic effect.
2. MATERIALS AND METHODS
2.1. Study sites and sediment sampling
The Bay of Kvarner (Croatia) is a relatively closed and isolated water body, located between
the Istrian peninsula and the northern Croatian coast (Fig. 1). The region is under the influence of
different types of industrial activities including oil processing, shipbuilding, cargo handling, among
others. The estimated annual load of industrial wastewater to the marine environment in the gulf of
Kvarner is approximately 560 million m 3, whereas the load of urban wastewater is approximately 23
million m3 (Cvitković, 2005). The yearly amount of untreated industrial wastewater that is being
released to the marine environment in the region is very large (182 million m 3), representing 33% of all
industrial wastewater generated in the region being released in the Bay of Kvarner.
For the purpose of this study six sampling sites, which are under the influence of different
types of pollution were chosen (S1–S6). Site S1 is a relatively unpolluted site used mostly for
recreational activities. Site S2 is the marina located in the western part of the Bay of Kvarner with a
capacity of 300 berths. Site S3 is the bulk cargo terminal, located in the region of Bakar bay, 13 km
from the city of Rijeka, used mainly for coal, iron and bauxite ore handling. Site S4 is the shipyard, the
second largest in Croatia. Site S5 is the port of Rijeka, one of the biggest ports in the Adriatic and also
the most important port in Croatia, whose main activities include e.g., loading, unloading,
warehousing, transportation of general cargo, timber, bulk cargo. Site S6 is the Mlaka oil refinery
which mainly produces lubricant oils, paraffin, fuel oil and tar, and is the largest producer of these
goods in Croatia (Traven et al., 2008). Sediments have previously been granulometrically classified as
sand (S1), sandy silt (S2) and silty sand (S3-S6) (Traven et al. 2008).
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The surface layer of sediments (0-5 cm) has been collected by SCUBA diving. The sediments
were transferred to the Department of Environmental Medicine (Medical faculty, University of Rijeka),
within 1 hour after sampling, frozen in polyethylene bags and stored at – 20oC until further processing.
The content of priority pollutants in the sediment samples has been previously determined by
Traven and co-workers (2008). According to the total content of PAH the sediment samples analysed
in this study were classified into two categories: as highly contaminated (S3, S4, S5 and S6) and
slightly contaminated sediments (S1 and S2). Regarding the content of polychlorinated biphenyls
(PCBs) the sediments at sampling stations S4 and S5 were polluted with PCBs at concentrations that
could cause some detrimental effects. With respect to heavy metal pollution, all heavy metals
analysed (Pb, Hg, Cd, Cu and Zn) were detected in all samples, with S3, S4, S5 and S6 sites being
more polluted than sites S1 and S2.
2.2. Sediment extracts preparation
Sediment samples (2 g) dried for 7 days at room temperature were extracted in an ultrasonic
bath at 25°C for 30 min with 15 mL of DCM/methanol (v:v – 2:1) as a solvent. A mixture of
DCM/methanol was used in order to extract a wide range of organic pollutants. The extract was then
filtered through a glass fibre filter, evaporated on a rotary evaporator and the dry residue was
dissolved in 1 mL of dimethyl sulfoxide (DMSO).
Sediment collected at the site S6 (Mlaka oil refinery) was additionally extracted using 3
different solvents (cyclohexane, DCM and methanol). Each time the sediments were extracted in an
ultrasonic bath at 25°C for 30 min with 15 mL of solvent.
2.3. PLHC-1 cell line culturing and exposure
PLHC-1 cells were maintained in 25 cm 2 flasks at 30°C in DMEM/F12 media (Invitrogen,
Carlsbad, USA) containing 5% of foetal bovine serum (FBS) (Invitrogen, Carlsbad, USA). The cells
were subcultured every 3–5 days at a split ratio of 1:3.
For the assessment of genotoxicity cells were grown in 24 well plates in 1 mL of medium for
24 h to allow for the complete cell attachment. The medium was then removed and replaced with 1 mL
of medium containing serial dilutions of the tested toxicant (0.1-10 µM for B[a]P and 10-1000 µM for
EMS) or sediment extract (0.08 – 20 mg/mL). Maximal DMSO concentration in the assay was 1%.
Non-exposed cells were used as a control and additional three replicas were exposed to medium
containing 1% DMSO in order to exclude its influence on DNA damage. Exposure was carried out in
triplicates for each test concentration used at 30°C for 24 h. After the incubation cells were washed
with 1 mL of phosphate buffer saline (PBS), trypsinized and processed for the Comet assay. Cell
viability was evaluated by the trypan blue exclusion assay and was above 95% for all tested
concentrations.
2.4. The Comet assay
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The alkaline Comet assay (single cell gel electrophoresis assay) was performed according to
the basic procedure of Singh et al. (1988) with slight modifications. Fifty μL of cell suspension was
mixed with fifty μL of 0.5% low melting point (LMP, Sigma-Aldrich) agarose and placed on a 1%
normal agarose precoated microscope slides. After solidifying for 3 min at 0°C, a third layer of 0.5%
LMP agarose was added and left to solidify as described. The cells were lysed in freshly made lysing
solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 10% DMSO, 1% Triton X-100, pH 10), for 1
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hour at 4 C. After rinsing with redistilled water, the slides were placed on the horizontal gel box,
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Electrophoresis was run in the same buffer at 25 V (0.83 V/cm) at 300 mA for 20 min at 4C. After
covered with the cold alkaline buffer (0.3 M NaOH, 1 mM EDTA, pH>13) and left for 20 min.
electrophoresis the slides were neutralized in a cold neutralization buffer (0.4 M Tris-HCl, pH 7.5), 2x5
min, fixed in methanol:acetic acid (3:1) for 5 min and stored in the dark at room temperature. Prior to
examination, the slides were rehydrated and stained with 10 µg/mL ethidium bromide and examined
using a Zeiss Axioplan epifluorescence microscope. Per each replica (per concentration) single slide
was prepared and per every slide at least 50 cells were examined. The extent of DNA migration was
determined as a percentage of DNA in the tail (% tDNA) using an image analysis system Komet 5,
Kinetic Imaging Ltd.
2.5. Statistical analysis
Mean values of the DNA damage for each group were calculated based on the mean of each
replica within a group and data presented as mean and corresponding SEM. Statistical analysis was
performed by the Mann–Whitney U-test. Level of significance reported: P ≤ 0.05. The induction factor
(IF) was calculated by dividing the mean value of % tDNA at each concentration by the mean value of
corresponding negative control. We also applied the Concentration dependent induction factor (CDI)
developed by Seitz and co-workers (2008). The CDI is a simple index that integrates all the important
data, providing a basis for a general comparison of the genotoxic potential in the Comet assay. The
CDI integrates all the concentrations and respective induction factors and is calculated according to
the following equation:
n
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CDI  
i 1
IFi
ci
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IFi = induction factor of the concentration i; ci = concentration i; n = n concentrations.
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3. RESULTS
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To allow for comparison of our data with data from other authors we have calculated CDI for the 4
concentrations ranging from 2.5 to 20 mg/mL.
3.1. Comet assay after exposure to model genotoxic compounds
Results of the Comet assay on PLHC-1 cell line after exposure to B[a]P and EMS showed a
statistically significant increase in DNA damage in a concentration dependent fashion (Fig 2a;2b). The
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highest DNA damage was observed at the highest toxicant concentration used for both B[a]P and
EMS with mean values of % tDNA 9.49 and 9.94 respectively.
3.2. Comet assay after exposure to marine sediment extracts
For all tested extracts, the Comet assay on PLHC-1 cells showed genotoxic effects in a
concentration dependent fashion (Fig 3a). Strong genotoxicity was detected in sediments from
locations S3, S5 and S6, with the highest percentage of DNA damage induced by the sediment from
the location S6 at the concentration of 1.25 mg/mL (9.53% tDNA). Other three sites (S1, S2, S4)
induced lower genotoxicity and the site S1 was the site that showed the lowest level of DNA damage
among all investigated sites.
3.2.1. Induction factor of sediment extracts
For most samples the maximum induction factor was recorded at the highest tested
concentration (20 mg/mL) with the exception of extracts from the sites S5 and S6 which induced the
highest genotoxicity at the concentrations 10 mg/mL and 1.25 mg/mL respectively (Fig 3b). Extracts
from the site S1 showed the lowest genotoxic effects with the maximum IF of 1.84, followed by
extracts from the sites S2 (2.07) and S4 (2.11). The strongest induction of DNA damage was recorded
for the extracts from sites S3, S5 and S6 and among them the highest IF was recorded for the extract
from the location S6 (3.68). The maximum IFs for extracts from the sites S3 and S5 were 3.61 and
3.21, respectively.
3.2.2. Concentration dependent induction factor of sediment extracts
Fig 3c shows the results calculated as CDI factors for all locations. There is a good correlation
between the calculated CDI factors and IF for the sites with the stronger genotoxicity (S3, S5 and S6)
confirming that the sample from the location S6 induced the highest genotoxic effect (CDI value 2.27).
CDI values for the samples from sites S3 and S5 had similar values (1.89 and 1.92 respectively).
Extracts from the sites S1, S2 and S4 had CDI values of 1.08, 1.35 and 1.42, respectively.
3.3. Comet assay after exposure to sediment extracts from site S6 obtained with different solvents
For all extracts of the sediment from the site S6 – oil refinery a positive dose-response
relationship in genotoxicity was observed (Fig 4a). The extract obtained with DCM/methanol induced
the strongest DNA damage, with the maximum value of tDNA recorded at the concentration 1.25
mg/mL (9.53%). A strong genotoxicity was also noticed for the extract obtained by cyclohexane (with
the maximum 8.75% tDNA at the concentration 20 mg/mL). Lower effect was observed with the
solvent DCM while the extract obtained with the methanol exhibited the lowest level of DNA damage.
3.3.1. Induction factor of sediment extracts from site S6 obtained with different solvents
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Fig 4b shows the results of induction factor for different solvents used. Extracts obtained with
methanol showed the lowest genotoxic effects with the maximum IF of 1.93, followed by extracts
obtained with DCM and cyclohexane with the maximum IF of 2.57 and 3.38 respectively. The
strongest induction of DNA damage was recorded for the extract obtained with DCM/methanol with a
maximum IF of 3.68. While maximal values of IF for a specific solvent were reached at the highest
concentration of sediment extract (20 mg/mL) for cyclohexane and methanol, for the DCM/methanol
and DCM the maximum IF were reached at the concentrations 1.25 mg/mL and 10 mg/mL
respectively.
3.3.2. Concentration dependent induction factor of sediment extracts from site S6 obtained with
different solvents
The results of CDI and IF for all solvents paralleled each other and confirmed that the sample
extracted with DCM/methanol induced the highest genotoxic effect (CDI value 2.27), followed by the
sample extracted with cyclohexane (CDI value 2.12). CDI values for the samples extracted with DCM
and methanol were similar and were 1.39 and 0.99 respectively.
4. DISCUSSION
The results of this study with model compounds and sediment extracts show that the
genotoxic compounds can easily be detected by the means of the Comet assay in the PLHC-1 fish cell
line. Exposure of cells to a model genotoxic agents (B[a]P, EMS) showed a significant increase in
DNA damage in a concentration dependent manner. DNA damage in PLHC-1 cells using the model
agent B[a]P showed a statistically significant increase of % tDNA at roughly the same concentration as
in rainbow trout hepatocytes (Devaux et al., 1997). Regarding the effect of EMS, it has been
previously demonstrated that concentrations lower than 1.5 µg/mL (12 µM) can induce chromosomal
damage in human lymphoblastoid cells (Doak et al., 2007). These results confirm our findings since
we have observed statistically significant DNA damage already at the lowest EMS test concentration
(10 µM).
All tested samples also induced genotoxic effects in the PLHC-1 cell line in a concentration
dependent manner. In general, the results of the Comet assay correlated well with the concentrations
of priority pollutants determined at each location. Higher level of DNA damage was noticed for
sediments from sites which are considered to be polluted (S3, S4, S5 and S6) while sites mostly used
for recreational purposes (S1) and marina (S2) demonstrated lower genotoxicity. However, certain
discrepancy between the concentration of priority pollutants and level of DNA damage was observed
for the site S3. Although having relatively lower amount of priority pollutants compared to the samples
S4 and S5, sample S3 exhibited stronger genotoxicity which could be due to the other genotoxic
compounds present in the sediment. Our results are in good correlation with the results obtained by
Traven et al. (2008) who performed the research using sediment samples from the same locations
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with the objective to compare the relationship between the concentration of priority pollutants and the
ability of sediment extracts to induce CYP1A in the PLHC-1 cell line. A similar gradation of toxicity of
the tested samples was observed in both studies with the sediment from the site S6 (oil refinery)
showing the strongest negative influence and having also the highest levels of priority pollutants. From
the priority pollutants detected in the sediment only PAHs and PCBs could be present in the sediment
extract due to the extraction procedure, indicating that those substances may contribute to the
observed genotoxicity. Similar conclusion was reached in the study done by Cachot and colleagues
(2006) where authors revealed a strongly significant relationship between SOS Chromotest responses
and the levels of PAHs. Strong correlation between the concentration of PAHs and PCBs in the
sediment and DNA strand breaks has been noticed in laboratory exposed Senegalese sole (Solea
senegalensis) as well (Costa et al. 2008). However, considering the fact that the sediment extracts are
complex mixtures of many contaminants the influence of non priority pollutants to the observed
genotoxicity can not be excluded. In a study performed by Kosmehl and colleagues (2007) moderate
pollution of sediments with heavy metals and priority PAHs could not be related to the major fraction of
genotoxic effects measured by the Comet assay with zebrafish embryos. The authors discussed that
high levels of DNA fragmentation have either been induced by effects of additional pollutants, or a
chemical analysis restricted to priority pollutants fails to address the necessary pollutant spectrum for
genotoxicity in terms of DNA strand breaks.
Comparisons of the results of the Comet assay carried out by different authors are often
confounded by differences in protocols and parameters measured (tail moment, tail length, % tail
DNA). For this reason, the results of different studies are compared using the induction factor (IF) and
CDI (taking into account 4 sediment dilutions, as implemented by Seitz et al., 2008). In this study the
sediment from the site S6 exhibited the strongest genotoxic effect at the concentration of 1.25 mg/mL
(IF 3.68) what is comparable to the results obtained by Rocha and co-workers (2009) who studied the
genotoxic potential of Tietê River sediments (São Paolo, Brazil) by means of the Comet assay on
RTL-W1 fish cell line after exposure to the sediment extracts. Although a direct comparison of the
results is not possible since they used different cell cultures and measured a different endpoint in the
Comet assay (tail moment), a general comparison can be made. The authors described site Billings
(São Paolo city region) as the site with strongest genotoxic influence on targeted cells and reported an
induction factor of 3.8 at the sediment extract concentration of 1.5 mg/mL. Furthermore, the sediment
collected from the reference site near the spring of the Tietê River exhibited IF of 1.4 at the
concentration 25 mg/mL which is comparable to our results for reference site S1 where maximum
genotoxicity was obtained at the concentration of 20 mg/mL (IF 1.84).
CDI values in our study were calculated based on 4 concentrations as used in the study done
by Seitz et al. (2008) and we noticed similar values for the site S6 (2.27) as for Ehingen on Danube
(2.75) which was classified as strongly toxic in another research done by Keiter et al. (2009).
In order to determine the contribution of the polar and non-polar contaminants responsible for
the observed toxic effects we have tested several solvents; methanol as a polar solvent, cyclohexane
and DCM as non-polar solvents as well as a solvent mixture DCM/methanol for the extraction of the
sediment from the highly polluted site S6. Our results showed that the strongest genotoxicity was
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observed for the sediment extracted with DCM/methanol, which extracts chemicals with a wide range
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polluted sites. Some previous studies reported high correlation of genotoxicity data between in vitro
of physical–chemical properties (Chen and White, 2004). In general we have noticed that non-polar
solvents yielded stronger genotoxicity than the more polar one. A similar conclusion was reached by
Vahl et al. (1997) in their study of the toxicity of sediments from the Elbe River (Germany). They
reported that less polar extracts (e.g. toluene) yielded mutagenic potency values measured with the
Ames test (TA98 with and without S9) which were higher than the methanol extract values. On the
other hand several other studies reached the opposite conclusions. The results obtained for samples
from the coastal Adriatic sediments (Croatia) contaminated with industrial and municipal waste water
showed that the detectable mutagenic activity (TA98 with S9) is primarily attributable to polar
compounds (Picer et al., 2001). Similar results were obtained in studies of sediments (TA 98 and TA
100 with S9) from the Kanawha River (USA) (Waldron and White, 1989). Kammann et al. (2004)
investigated genotoxic potential of fractionated marine sediment extracts from the North and Baltic
Sea by means of the Comet assay on EPC cells. They reached the conclusion that polar fractions
induced genotoxic effects while no genotoxicity was measured in non-polar fractions containing PAHs.
Our results, obtained on sediment extracts from site S6, indicated the opposite, that the non-polar
component induces more DNA damage. It has been demonstrated that the sediment from this location
contains high amounts of PAHs (Traven et al. 2008) which have been argued as cause of genotoxic
effects in fish or fish cells detected with the Comet assay (Kammann et al., 2001; Akcha et al. 2003).
The discrepancy between the results obtained in different studies could be explained by differences in
PAHs composition in the sediment as well as differences in the presence and quantity of other
genotoxic substances.
Sediment bioassays are suitable to yield data with the respect to toxic effects in selected test
systems although the selected tests are conducted under laboratory conditions and usually can not be
applied under in situ conditions which encourage the use of integrated approaches in order to give
insight into the ecological state of sediments (Hollert et al., 2002). Even though the relevance of the
results obtained by the sediment extracts is not as ecologically important as the investigations
implementing native sediments or exposure in situ since they do not reflect the real environmental
conditions where many factors can influence the bioavailability and consequently the toxicity of the
tests and field studies (Rocha et al., 2009; Boettcher et al. 2010) confirming the usefulness of tests
implementing sediment extracts in DNA damage assessment.
5. CONCLUSION
The results of this study indicate that the Comet assay on the PLHC-1 cell line is a costeffective ecotoxicological tool for screening contaminated marine sediments for the presence of
genotoxic compounds. In addition, our data for the most polluted site (S6) point out that the observed
genotoxicity can be attributed mainly to non-polar compounds present in contaminated marine
sediments.
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ACKNOWLEDGMENTS
This study was made within the framework of the projects no. 119-0982934-3110 and 0620621341-0308 supported by the Ministry of Science, Education and Sports of the Republic of Croatia.
The authors are very grateful to Adam Maguire for the English revision of the manuscript.
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Figure captions
Figure 1. Sampling sites in the Bay of Kvarner, Croatia. S1, site used for recreational activities; S2,
marina; S3, bulk cargo terminal; S4, shipyard; S5, port; S6, oil refinery.
Figure 2. Results of the Comet assay on PLHC-1 cell line after exposure to B[a]P (a) and EMS (b) (*
statistical significance comparing to the control; P<0.05).
Figure 3. Genotoxic effects of DCM/methanol marine sediment extracts on PLHC-1 fish cell line after
24 hours exposure; a) Results of the Comet assay (* statistical significance comparing to the control;
P<0.05); b) Induction factor (IF) of DNA damage; c) Concentration dependant induction factor (CDI)
values.
Figure 4. Genotoxic effects of marine sediment extracts from the site S6 - oil refinery extracted with
various solvents on PLHC-1 fish cell line after 24 hours exposure; a) Results of the Comet assay (*
statistical significance comparing to the control; P<0.05); b) Induction factor (IF) of DNA damage; c)
Concentration dependant induction factor (CDI) values.
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