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Aroclor 1254 May Induce Common DNA Effects in Developing Paralichthys olivaceus Embryos and Larvae
Aroclor 1254 May Induce Common DNA Effects in Developing Paralichthys olivaceus Embryos and Larvae
Fisheries and aquatic sciences. 2014. Dec, 17(4): 461-469
Copyright © 2014, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licens (http://creativecommons.org/licenses/by-nc/3.0/)which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : March 03, 2014
  • Accepted : July 07, 2014
  • Published : December 31, 2014
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About the Authors
Eun Young Min
Institute of Fisheries Sciences, Pukyong National University, Busan 608-737, Korea
Ju Chan Kang
Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Korea
jxheart@nate.com
Abstract
Polychlorinated biphenyls (PCBs) are persistent pollutants in aquatic environments, often causing the decline or disappearance of wild populations. In this study, we used a random amplified polymorphic DNA (RAPD) assay to evaluate the effects on the genomic DNA of olive flounder embryo and larval stages of exposure to Aroclor 1254 at concentrations of 1, 5, 10, 20, and 40 μg/L. We compared RAPD fingerprints of exposed and non-exposed samples. Polymorphisms were revealed as the presence and/or absence of DNA fragments between the two samples. A dose-dependent increase in the number of polymorphic bands was observed with Aroclor 1254 treatment. Also, RAPD profiles of animals exposed to Aroclor 1254 exhibited an increase in the frequency values (FV) compared to the control. A phenogram constructed using neighbor-joining method indicated that genomic template stability in developing embryo and larval stages was significantly affected at ≥ 5 μg/L. This study suggested that DNA polymorphisms detected by RAPD analysis could be used as an investigative tool for environmental toxicology and as a useful biomarker in early life stages for the detection of potential genotoxicants.
Keywords
Introduction
Pollutants with genotoxic potential are of great concern to many ecotoxicologists. Once released, these agents have the capability not only to cause morbidity and/or mortality in exposed organisms, but also to affect biological diversity at both intra- and interspecies levels ( Atienzar et al., 1999 ). However, despite growing concerns over the presence of genotoxins in aquatic environments, adequately validated methods that can be used to evaluate genotoxicity and associated toxicity in aquatic organisms under environmentally relevant conditions are lacking. To examine genotoxic impacts, the three classic methods typically used are the micronucleus assay, the comet assay, and 8-oxoguanosine quantification. However, previous studies have demonstrated that in the eel, Anguilla anguilla , the micronucleus test was not sufficiently sensitive to detect aquatic pollution, nor was the comet assay sufficiently sensitive in mussel gills ( Sanchez-galan et al., 2001 ; Pruski and Dixon, 2002 ). Another difficulty linked to the micronucleus test and the comet assay is the necessity to experiment on isolated cells ( Cambier et al., 2010 ). The 8-oxoguanosine method is a sensitive technique for quantifying genotoxic impacts of xenobiotics, but it is a time-consuming and fastidious process ( Halliwell, 1999 ). However, remarkable disagreement remains concerning the values at steady state in liver tissue DNA due to the marked variability of the results obtained by various laboratories ( Halliwell, 1999 ; European Standards Committee on Oxidative DNA Damage (ESCODD), 2002 ).
Aroclor 1254, a commercial mixture of polychlorinated biphenyl (PCB) congeners, can be carcinogenic, clastogenic, and cause birth defects in both animals and humans ( Cogliano, 1998 ; Sargent et al., 1989 ). While PCBs, such as dioxin, are usually considered to be non-genotoxic carcinogens ( Whysner et al., 1998 ), there remains some uncertainly in this regard. The mechanisms by which PCBs exert their adverse effects are not fully understood and data on their genotoxic properties are controversial ( Ross, 2004 ). Silberhorn et al. (1990) reported that Aroclor 1254 had negative results in the majority of genotoxicity assays. However, Aroclor 1254 produced DNA single-strand breaks in an alkaline elution/rat hepatocyte assay ( Sina et al., 1983 ) and was clastogenic in dove embryos ( Peakall et al., 1972 ). Accordingly, in this study, the genotoxic effect of Aroclor 1254 has been assessed using a random amplified polymorphic DNA (RAPD)-based methodology.
RAPD is currently used for intra-populational polymorphism detection and also enables the detection of genotoxicity after pollutant contamination of animals ( Grayson et al., 1999 , 2000 ). Moreover, Atienzar and Jha (2006) suggested that RAPD is a reliable, sensitive, and reproducible assay, and after proper optimization, has the potential to detect a wide range of DNA damage as well as mutations and therefore can be applied to genotoxicity and carcinogenesis studies. In fact, RAPD has been successfully used to detect ‘DNA effects’ induced by copper, cadmium benzo[ a ]pyrene, and nonylphenol, mainly in aquatic systems ( Atienzar et al., 1999 , 2001 , 2002 , 2004 ; Atienzar and Jha, 2004 , 2006 ; Castano and Becerril, 2004 ; Cambier et al., 2010 ). The most significant advantages of the RAPD assay lie in its rapidity, applicability to any organism, and ability to detect a wide range of DNA damage and mutations, including point mutations and large rearrangements ( Atienzar et al., 2002 ). However, in these previous studies, exposure to genotoxins was conducted using concentrations higher than those generally found in environmentally polluted areas.
Some of the most persistent and ubiquitous contaminants are PCBs, although they are found only at low concentrations( Domingo and Bocio, 2007 ; Brown et al., 2006 ; Ross, 2004 ). Recently, the PCB contents of muscle tissues of marine fishes from nine coastal cities of East China were analyzed. The total PCB concentrations in all fish samples varied between 13.3 and 78.3 μg/kg lipid weight (lw), with an average of 35 ± 15 μg/kg lw (Xia et al., 2012). In our previous study on the toxicological effect of Arolcor 1254, the LC 50 values in olive flounder embryos and larvae were 50.92 μg/L and 3.08 μg/L, respectively, and exposure to concentrations as low as 1 μg/L disrupted the early developmental stages of the fish ( Min and Kang, 2013 ). An expert panel of the European Food Safety Authority (EFSA) concluded that we must improve our understanding of the environmental and human risks associated with PCBs due to their abundance in food and human tissues ( Stenberg and Andersson, 2008 ).
According to Koh et al. (2001) , the concentration of chlorinated compounds such as PCBs and dichlorodiphenyl trichloroethane (DDT) among the organic pollutants in Korean coastal environments was lower than in other developed countries. However, polycyclic aromatic hydrocarbons (PAHs), which are toxic compounds derived from oil spills, were present at higher concentrations in Korean coastal regions ( Koh et al., 2001 ). Due to their high persistence and strong lipophilic properties, PCBs have bioaccumulated in aquatic food webs. Although the water solubility of Aroclor 1254 is only 2.7 μg/L, PCBs have been classified as probable human carcinogens by the Environmental Protection Agency (EPA), based on substantial evidence of cancer in animals ( Walker, 1994 ). In fishes, PCBs such as Aroclor 1254 also are transported with lipoproteins into the yolk of developing oocytes ( Ungerer and Thomas, 1996 ). Increased concentrations of PCBs in fish embryos and ovaries have been correlated with decreased viability, embryo toxicity, and embryonic malformations ( Black et al., 1988 ; Weis and Weis, 1989 ; Min and Kang, 2013 ).
The development of such in vivo test systems is also essential to provide a scientific basis for comparing the relative risks of genotoxins ( Jha et al., 2000a , 2000b ). DNA damage induced in the early life-stage (ELS) of an organism inhibits its development toward adulthood, which might cause disturbances to the ecosystem along the food chain as well as serious economic loss in fisheries. Aquatic toxicologists generally consider ELS toxicity tests to be the most useful for risk assessment because the ELS is a sensitive stage of the fish life cycle during which many critical events take place over a very short span of time ( Von Westernhagen, 1988 ). During this phase, man-made pollutants may exert strong effects on the survival of fish embryos and larvae. Attractive features of this method are that it is a relatively simple bio-indicator, and that it provides good coverage, even for larger marine areas ( Cameron and Berg, 1992 ).
For environmental monitoring purposes, one of the major biological challenges has been to bring potential organisms into continuous routine culture. This is especially important when sound genetic analysis is to be carried out. Olive flounder Paralichthys olivaceus , are distributed among coastal areas of Korean and Japan. In Korea, this species is commonly raised in aquaculture, and it is important both in the market and on the table. In this study we chose the olive flounder because of its economic value as a food resource as well as the considerable ease in handling and procurement.
The aims of this study were to detect potential genotoxic effects induced by Aroclor 1254 at environmentally relevant dosages using an RAPD technique, and to confirm the potential for the use of embryos and larvae of the olive flounder P. olivaceus , in in vivo laboratory studies for biomonitoring of genotoxic risk evaluation.
Materials and Methods
- Experimental animals and water conditions
Fertilized eggs of the olive flounder P. olivaceus were obtained from hatcheries in Cheju Island, South Korea. The water quality parameters measured for the bioassays conducted were as follows: pH, 8.10 ± 0.2; salinity, 32.70 ± 0.4‰; dissolved oxygen, 6.74 ± 0.84 mg/L; chemical oxygen demand, 1.52 ± 0.008 mg/L. All experiments were conducted at a seawater temperature of 20 ± 0.5°C under a 12-h light/12-h dark cycle, over a 36- or 72-h period. Other pertinent test conditions for the acute toxicity tests are summarized in Table 1 .
Test conditions for the acute toxicity tests with olive flounderParalichthys olivaceusembryos and larvae
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Test conditions for the acute toxicity tests with olive flounder Paralichthys olivaceus embryos and larvae
- Chemicals
Aroclor 1254 was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Five solutions (1, 5, 10, 20, and 40 μg/L) were prepared by diluting a stock solution. The stock solution was prepared with seawater that had been filtered through a grade GF/C Whatman filter (Maidstone, UK) and UV-treated.
- Embryo and larval toxicity tests
The eggs obtained from the hatchery were approximately at the 8–10-h post-fertilization (blastula) stage. They were transferred directly into clean 500-mL glass beakers containing 0 (control), 1, 5, 10, 20, or 40 μg/L Aroclor 1254 for static exposure. Each Aroclor 1254 concentration (including the control) was carried out in triplicate beakers. Embryonic development was monitored every 3 h, until an endpoint at 40 h, or until hatching was complete. The beaker contents (Aroclor 1254 solutions) were replaced every 24 h throughout the toxicity test ( Table 1 ). Newly hatched larvae were exposed to identical Aroclor 1254 concentrations to maintain the conditions ( Table 1 ). The larvae were monitored every 6 h for up to 72 h.
At each time point during the embryo and larva toxicity tests, from each beaker, 50 organisms were collected, washed with UV-treated seawater, placed in phosphate-buffered saline (PBS), frozen in liquid nitrogen, and then stored at –70°C until subsequent analysis. The entire experiment was repeated in triplicate.
- DNA isolation and DNA profiling using RAPD
The genomic DNA (gDNA) of exposed test organisms was extracted using a commercial DNA purification kit (NucloGen Biothechnology, Korea). Briefly, the organisms were lysed using sodium dodecyl sulfate and DNA was extracted by proteinase K digestion, phenol:chloroform extraction, and ethanol precipitation. DNA concentration was measured using a Bio-photometer (Eppendorf AG, Germany). DNA stock solutions were diluted to 10 μg/mL in distilled water and stored at –70°C until analysis.
DNA profiles of P. olivaceus were generated with RAPD reactions performed in 20-μL reaction volumes as described previously ( Atienzar et al., 2000 ). The oligonucleotides AP1– AP8 were obtained from an oligo synthesis company (Bioneer Co., Korea; the sequences are provided in Table 2 ). DNA amplification was performed on a My Cycler™ thermal cycler (Bio-Rad, USA), and the amplification protocol was as follows: 5 min at 95°C , followed by 35 cycles of 1 min at 94°C , 2 min at 45°C, and 2 min at 72°C.
Sequence of the primers used for RAPD PCR in this study
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Sequence of the primers used for RAPD PCR in this study
Electrophoresis of RAPD reaction products were performed in 2% w/v agarose gels, using a Tris-Borate-EDTA buffer system (1X TBE; 90 mM Tris-base, 90 mM boric acid, and 2 mM EDTA) at 100 volts for 30 min. The gels were stained in a 1X TBE solution containing ethidium bromide (0.015% v/v) for at least 40 min. Gels were visualized under UV light, and photographed using a Gel-doc camera system (Gel-doc XR. Bio-Rad. USA). For comparison, a DNA molecular size marker (100-bp DNA Ladder, Bioneer, Co. Korea) was used.
- Data analysis
The RAPD method is relatively new and there is no one standard statistical method used routinely for determining genetic diversity. For comparison, we used neighbor-joining analysis and frequency values (FV). Neighbor-joining analysis ( Saitou and Nei, 1987 ), in which genetic distances are computed for all pairs of individuals, was performed using the NEIGHBOR program in PHYLIP 3.5c ( Felenstein, 1993 ) to generate an unrooted phenogram. A phenogram was constructed based on all individuals examined in this study. In the phenogram, samples from the various concentration groups with similar genotypes can be identified through clustering. Also, in this study, the FVs obtained from calculating band counts of new appearances or disappearances (i.e., mismatched bands compared to the control) in the RAPD profiles using each primer were calculated as follows:
  • Frequency values of contamination indicative bands (FV) = (ΣNA+ ΣND)/NP
  • ΣNA: the sum of the total number of newly appeared bands in each primer RAPD profile
  • ΣND: the sum of the total number of disappeared band in each primer RAPD profile
  • NP: the total number of primers used in the RAPD profile
The frequency values of contamination indicative profiles (FV) were calculated using a modification of a protocol outlined by Theodorakis and Shugart (1997) . Treatment groups were compared to the control using a one- or two-way analysis of variance (ANOVA). If the ANOVA was significant ( P < 0.05), a Duncan’s multiple comparison test was used to identify means that were similar and those that were significantly different. For continuously recorded parameters, the median effective concentration (EC 50 ) values of FV (i.e., the statistically derived concentration of a substrate in an environmental medium expected to produce a certain effect in 50% of test organisms in a given population under a defined set of conditions), including upper and lower 50% confidence limits, were determined by Probit analysis using the SPSS/PC+ statistical package.
Results and Discussion
The continuous presence of genotoxic chemicals in aquatic environments is of major concern due to their effects on the health of aquatic biota ( Jha, 2004 ). DNA integrity is one of the biomarkers of pollution, and therefore much effort has been focused on finding rapid, sensitive methods for obtaining information on potential genotoxicity ( Sarkar et al., 2006 ). In fact, several studies have used PCR-based techniques to assess genetic variation and changes in aquatic biota exposed to different contaminants ( Theodorakis and Shugart, 1997 ; Nadig et al., 1998 ; Ma et al., 2000 ). In addition, it has been suggested that RAPD assays can detect mutations only if they occur in at least 2% of the DNA ( John and Kortenkamp, 2000 ).
- RAPD DNA profiles of embryos and larvae under Aroclor 1254 stress
In this study, RAPD analyses were performed on gDNA pooled from 50 individuals of uncontaminated or Aroclor 1254-contaminated embryos or larvae. gDNA was pooled from 50 individuals to suppress intra-populational genetic polymorphisms that could potentially be revealed by RAPD. Eight different random primers were screened to discriminate controls from contaminated embryos and larvae. Of the eight primers tested, only four gave specific and stable results, and RAPD profiles generated with primers P2 and P6 were especially highly polymorphic. To determine the diversity of organisms exposed to Aroclor 1254, profiles were sorted based on the RAPD fragments at each exposure time ( Fig. 1 ). The RAPD profiles showed substantial differences between unexposed and exposed animals, with apparent changes in the number, size, and intensity of amplified DNA fragments. Asterisks and the letter “d” on the RAPD profile gels of exposed groups indicate some of the obvious differences from the control ( Fig. 1 ).
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RAPD profiles obtained with the primers 2 (C), 6 (A, B and E) and 7 (D). DNA extracts form Paralichthys olivaceus embryos (A, B, and C) and larvae (D and E) exposed to various concentrations of Aroclor 1254; Control (C), 1, 5, 10, 20 and 40 μg/L for 12 (A), 24 (B), 36 (C), 60 (D) and 72 (E) hours. Selected changes are indicated by a symbol (*, variation in band intensities and appearance of a new band; d, disappearance of a band) in comparison to control patterns in profiles. The 100 bp DNA ladder (M) was used to as DNA molecular weight marker.
- Analysis of data generated by RAPD profiling
Neighbor-joining analysis assigns a genotype to each examined fish based on RAPD profiles generated by all analyzed primers and further reveals genetic relationships among all examined fish ( Nadig et al., 1998 ). Because there was genetic differentiation among the treatment groups, a phenogram of genetic distance among the groups may be useful in delineating water quality ( Fore et al., 1995 ). In this study, Fig. 2 is a phenogram generated using all primers and shows that the embryos were divides into two groups, designated here as the affected cluster (A), containing embryos affected by Aroclor 1254, and the non-affected cluster (NA). Interestingly, the number of the exposed group belonging to cluster (A) increased in an exposure-time-dependent manner in the embryo stage, increasing from two groups (20 and 40 μg/L) at 12 h, to four groups (5, 10, 20 and 40 μg/L) at 36 h ( Fig. 2 ). In the larval stages, the phenogram also indicates two genetic clusters: cluster (NA) (1 and 5 μg/L), was more similar to the control than to cluster (A) (10 and 20 μg/L). The results demonstrate that the genotypes of embryos and larvae were highly variable, indicating that the genetic diversity of Aroclor 1254-contaminated groups can be readily analyzed by RAPD.
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Phenogram constructed using RAPDPLOT Neighbor joining method incorporating all primers in embryos (I, II, III) and larvae (IV, V) from control and different Arclor 1254 concentrations; 1, 5, 10, 20 and 40 μg/L. Two major clusters of genotype (NA and A) indicates smaller clusters within each of these major groups.
The phenetic numerical analysis of RAPD profiles is the most popular way to analyze RAPD in ecotoxicology studies and has been used to determine genetic diversity in nat-ural populations exposed to a variety of pollutants ( Ross et al., 2002 ; Turuspekov et al., 2002 ). On a phenetic basis, the diversity is estimated by comparing the absence/presence of RAPD bands in exposed and non-exposed individuals (i.e., similarity analysis), or by counting their RAPD band numbers and considering band abundances ( Maldonado et al., 2003 ). Modifications in band intensity and lost bands are likely to be due to one or a combination of the following events: (1) changes in oligonucleotide priming sites due to genomic rearrangements (more likely) and/or point mutations and DNA damage in the primer binding sites (less likely), and (2) interactions of DNA polymerase in organisms with damaged DNA ( Liu et al., 2005 ). Thus, the FVs of these DNA polymorphisms could be used for the detection of genotoxic effects. In this study, changes in RAPD profiles were expressed as increases in FV (a measure reflecting the obvious changes in DNA patterns generated from toxicant-exposed organisms) in relation to profiles obtained from control organisms ( Fig. 3 ).
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Frequency values of contamination indicative bands compared to control in the RAPD profiles from embryos and larvae exposed to different Aroclor 1254 concentration for different exposure periods. Values (mean ± standard error) with the same superscript letter are not significantly different (P>0.05).
The EC 50 values of FVs determined for embryos and larvae exposed to different concentrations of Aroclor 1254 are shown in Table 3 . FVs significantly increased in a dose-dependent manner in developing embryos exposed to Aroclor 1254, but in the larval stage, there was no significant difference among the concentration groups ( Fig. 3 ). Although FVs in larvae were not significantly different between the groups, a high FV was sustained until 72 h. This result suggests that during ELS, changes to gDNA stability may be an effect of the genotoxic potential of Aroclor 1254. This result also suggests that gDNA stability in the larval stage may be more sensitive than in the embryonic stage. As mentioned in our previous study ( Min and Kang, 2013 ), our results demonstrate that Aroclor 1254 disrupted the early development stages of olive flounder, with higher toxicity in the larvae than in the developing, unhatched embryos.
The value of EC50of the frequency values (FV) determined by Probit analysis in olive flounderParalichthys olivaceusembryos and larvae exposed to Aroclor 1254
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The value of EC50 of the frequency values (FV) determined by Probit analysis in olive flounder Paralichthys olivaceus embryos and larvae exposed to Aroclor 1254
Several studies have used ELS marine organisms to detect genomic instability caused by pollutants ( Jha et al., 2000a , 2000b ). Atienzar et al. (2002) suggested that 4-n-Nonylphenol and 17β-estradiol induced DNA effects in larvae of the barnacle Elminius modestus , and these specific modifications in RAPD patterns may arise as a consequence of hot spot DNA damage, DNA adduct formation, and point mutations or genomic rearrangements. Jha et al. (2000a) demonstrated that tributyltin oxide induced potential genotoxic effects in the ELS of the marine mussel Mytilus edulis . These previous results are in agreement with our study. These data indicate that it may be possible to relate DNA damage from the pooled embryos and larvae to similar damage in DNA isolated from the organs, that is, the liver, gills, and blood cells, of individual fish. In this study, using a genotoxicity assay in vivo based on the larval stage of olive flounder, we have evaluated the potential genotoxic hazard of Aroclor 1254. The results obtained from this study provide circumstantial evidence of the mechanisms mediating some of the DNA changes. The developmental (ELS) toxicity results in our previous study confirmed that Aroclor 1254 caused toxic responses in the larvae of olive flounder ( Min and Kang, 2013 ). The developmental toxicity results complemented the genotoxic response at lower levels of biological organization, as more sensitive and robust end points. The cytotoxic effects can be detected alterations on a specific mechanism at DNA level using the cytogenetic assays, which would have direct implications for the developmental, and subsequently reproductive, success of the exposed organism ( Jha et al., 2000b ).
PCBs were not genotoxic in the bacterium Salmonella typhimurium or V79 Chinese hamster cells in vitro ; only in rat liver did lower chlorinated congeners lead to covalently modified macromolecules including proteins and DNA ( Knerr and Schrenk, 2006 ). However, in in vitro tests using the fish cell line RTG-2 from the rainbow trout, the comet assay and the micronucleus test showed clear genotoxic damage after PCB exposure ( Marabini et al., 2011 ). Several mechanisms could account for the genotoxic potential of PCBs. The cause of the genotoxic damage from PCBs may be oxidative stress, which could give rise to reactive oxygen species (ROS) production, lipid peroxidation, loss of glutathione (GSH) content and reduced superoxide dismutase (SOD) levels, with significant amounts of DNA oxidized bases ( Marabini et al., 2011 ). A previous study has linked PCBs such as Aroclor 1254 and their hydroxylated metabolites (OH-PCBs) with ROS production ( Strathmann et al., 2006 ). The PCBs, metabolized by the CYP450 system, reportedly formed dihydroxylated metabolites such as catechols and hydroquinones. These were oxidized to semiquinones, which can damage DNA directly ( Schlezinger et al., 2006 ). Previous studies have also demonstrated that PCBs cause double-strand DNA breaks in yeast and bluegill sunfish, and have suggested that this is secondary to oxidative stress ( Theodorakis et al., 1992 ; Appelgren et al., 1999; Tharappel et al., 2002).
In the field of “genetic-ecotoxicology” or “eco-genotoxicology”, there are few well-validated tools for linking genetic damage to end points of direct importance to the ecological viability of marine populations ( Anderson et al., 1994 ; Jha et al., 2000a ). This study will provide a useful strategy in which first, RAPD analysis can be used as a screening method for potential genotoxic effects and, second, phenetic numerical analysis can be applied to measure DNA instability resulting from pollutants. These techniques are therefore being adopted as sensitive methods for the detection of induced genetic damage at the molecular level in both embryos and larvae of aquatic organisms.
In conclusion, our results indicate that Aroclor 1254 increased DNA damage in the embryo and larval stages (ELS) of olive flounder, as assessed by RAPD analysis. We suggest that DNA polymorphisms detected using RAPD analysis could be used for investigation of environmental toxicology and as a useful biomarker in an early warning system. In addition, we established this method under laboratory conditions using ELS and provided evidence of a reproducible and sensitive model for the detection of genotoxicants.
References
Anderson S , Sadinski W , Shugart L , Brussard P , Depledge M , Ford T , Hose J , Stegeman J , Suk W , Wirgin I , Wogan G 1994 Genetic and molecular ecotoxicology: A research framework Environ Health Perspect 102 3 - 8
Atienzar FA , Conradi M , Evenden AJ , Awadhesh NJ , Depledge MH. 1999 Qualitative assessment of genotoxicity using random amplified polymorphic DNA: comparision of genomic template stability with key fitness parameters in Daphia magna exposed to Benzyo(a)pyrene Environ Toxicol Chem 18 2275 - 2282
Atienzar FA , Evenden AJ , Jha A , Savva D , Depledge M 2000 Optimized RAPD analysis generates high-quality genomic DNA profiles at high annealing temperature Bio Techniques 28 52 - 54
Atienzar FA , Cheung VV , Jha AN , Depledge MH 2001 Fitness parameters and DNA effects are sensitive indicators of copper induced toxicity in Daphnia magna Toxicol Sci 59 241 - 250
Atienzar FA , Billinghurst Z , Depldge MH 2002 4-n-Nonylphenol and 17-β estradiol may induce common DNA effects in developing barnacle larvae Environ Poll 120 735 - 738
Atienzar FA , Jha AN 2004 The random amplified polymorphic DNA (RAPD) assay to determine DNA alterations, repair and transgenerational effects in B(a)P exposed Daphnia magna Mutat Res 552 125 - 140
Atienzar FA , Jha AN 2006 The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review Mutat Res 613 76 - 102
Black DE , Phelps DK , Lapan RL 1988 The effect of inherited contamination on egg and larval winter flounder, Pseudopleuronectes americanus Mar Environ Res 25 45 - 62
Brown FR , Winkler J , Visita P , Dhaliwal J , Petreas M 2006 Levels of PBDEs, PCDDs, PCDFs and coplanar PCBs in edible fish from California coastal waters Chemosphere 64 276 - 286
Cambier S , Gonzalez P , Durrieu G , Bourdineaud JP 2010 Cadmium-induced genotoxicity in zebrafish at environmentally Ecotoxicol. Environ Safe 73 312 - 319
Cameron P , Berg J 1992 Morphological and chromosomal aberrations during embryonic development in dab (Limanda limanda) Mar Ecol Prog Ser 91 163 - 169
Castano A , Becerril C 2004 In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line Mutat Res 552 141 - 151
Cogliano VJ 1998 Assessing the cancer risk from environmental PCBs Environ. Health Perspect 106 317 - 323
Danis B , Wantier P , Flammang R , Pernet PH , Chambost-Manciet Y , Coteur G , Warnau M , Dubois PH 2006 Bioaccumulation and effects of PCBs and heavy metals in sea stars (Asteria rubens L.) form the North Sea: a small-scale perspective Sci Total Environ 356 275 - 289
Domingo JL , Bocio A 2007 Levels of PCDD/PCDFs and PCBs in edible marine species and human intake: a literature review Environ Int 33 397 - 405
2002 Comparative analysis of baseline 8-oxo-7, 8-dihydroguanine in mammalian cell DNA, by different methods in different laboratories: an approach to consensus Carcinogenesis 23 2129 - 2133
Felenstein J 1993 PHYLIP (Phylogeny Inference Package) version 3.5c Department of Genetics, University of Washington Seattle, USA Produced and distributed by author
Fore SA , Guttman SI , Bailer AJ , Altfater DJ , Counts BV 1995 Exploratory analysis of population genetic assessment as a water quality indicator: I. Pimephales notaus Ecotoxicol Environ Safe 30 24 - 35
Grayson TH , Cooper LF , Atienzar FA , Knowles MR , Gilpin ML 1999 Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations Appl Environ Microbiol 65 961 - 968
Grayson TH , Atienzar FA , Alexander SM , Cooper LF , Gilpin ML 2000 Molecular diversity of Renibacterium salmoninarum isolates determined by randomly amplified polymorphic DNA analysis Appl Environ Micorbiol 66 435 - 438
Halliwell B 1999 Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition Mutat Res 443 37 - 53
Jha AN , Hagger JA , Hill SJ 2000 Tributyltin induces cytogenetic damage in the early life stages of the marine mussel, Mytilus edulis Environ Mol Mutat 35 434 - 350
Jha AN , Cheung VV , Foulkes ME , Hill SJ , Depledge MH 2000 Detection of genotoxins in the marine environment: adoption and evaluation of an integrated approach using the embryo-larval stages of the marine mussel, Mytilus edulis Mutat Res 464 213 - 228
Jha AN 2004 Genotoxicological studies in aquatic organisms: an overview Mutat Res 552 1 - 17
Jones C , Kortenkamp A 2000 RAPD library fingerprinting of bacterial and human DNA: applications in mutation detection Teratog Carcinog Mutagen 20 49 - 63
Knerr S , Schrenk D 2006 Carcinogenicity of “Non-dioxinlike” Polychlorinated biphenyls Crit Rev Toxicol 36 663 - 694
Koh CH , Kim GB , Maruya KA , Anderson JW , Jones JM , Kang SG 2001 Induction of the P450 reporter gene system bioassy by polycyclic aromatic hydrocarbons in Ulsan Bay (South Korea) sediments Envrion Pollut 111 437 - 445
Liu W , Li PJ , Qi XM , Zhou QX , Zheng L , Sun TH , Yang YS 2005 DNA changes in barley (Hordeum vulgare) seedlings induced by cadmium pollution using RAPD analysis Chemosphre 61 158 - 167
Ma XL , Cowles DL , Carter RL 2000 Effect of pollution on genetic diversity in the bay mussel Mytilus galloprovincialis and the acorn barnacle Balanus glandula Mar Environ Res 50 559 - 563
Maldonado San Martin AP , Adamec L , Suda J , Mes THM , Storchova H 2003 Genetic variation within the endangered species Aldrovanda vesiculosa (Droseraceae) as revealed by RAPD analysis Aquat Bot 72 159 - 172
Marabini L , Calo R , Fucile S 2011 Genotoxic effects of polychlorinated biphenyls (PCB 153, 138, 101, 118) in a fish cell line (RTG-2) Toxicol Vitro 25 1045 - 1052
Min EY , Kang JC 2013 Toxicological effects of Aroclor 1254 on the embryonic development of the olive flounder Paralichthys olivaceus Fish Aquat Sci 16 253 - 260
Nadig SG , Lee KL , Adams SM 1998 Evaluation of alterations of genetic diversity in sunfish populations exposed to contaminations using RAPD assay Aquat Toxicol 43 163 - 178
Peakall DB , Lincer JL , Bloom SE 1972 Embryonic mortality and chromosomal alterations caused by Aroclor 1254 in ring doves Environ. Health Perspect 1 103 - 104
Pruski AM , Dixon DR 2002 Effects of cadmium on nuclear integrity and DNA repair efficiency in the gill cells of Mytilus edulis L Aquat Toxicol 57 127 - 137
Ross K , Cooper N , Bidwell JR , Elder J 2002 Genetic diversity and metal tolerance of two marine species: a comparison between populations from contaminated and reference sites Mar Pollut Bull 44 671 - 679
Ross G 2004 The public health implications of PCBs in the environment Ecotoxicol. Environ. Safe 59 275 - 291
Saitou N , Nei M 1987 The neighbor-joining method: a new method for constructing phylogenetic trees Mol Biol Evol 4 406 - 425
Sanchez-Galan S , Linde AR , Ayllon F , Garcia-Vazquez E 2001 Induction of micronuclei in eel (Anguilla anguilla L.) by heavy metals Ecotoxicol Environ Saf 49 139 - 143
Sargent L , Roloff B , Meisner L 1989 In vitro chromosome damage due to PCB interactions Mutat Res 224 79 - 88
Sarkar A , Ray D , Shrivastava AN , Sarker S 2006 Molecular biomarker: their significance and application in marine pollution monitoring Ecotoxocology 15 333 - 340
Schlezinger JJ , Struntz WDJ , Goldstone JV , Stegeman JJ 2006 Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar polychlorinated biphenyl congeners Aquat Toxicol 77 422 - 432
Silberhorn EM , Glauert HP , Robertson LW 1990 Carcinogenocity of poly-halogenated biphenyls: PCBs and PBBs Crit Rev Toxicol 20 440 - 496
Sina JF , Bean CL , Dysart GR , Taylor VI , Bradley MO 1983 Evaluation of the alkaline elution/rat hepatocyte assay as a predictor of carcinogenic/mutagenic potential Mutat Res 113 357 - 391
Stenberg M , Andersson PL 2008 Selection of non-dioxin-like PCBs for in vitro testing on the basis of environmental abundance and molecular structure Chemosphere 71 1909 - 1915
Strathmann J , Schwarz M , Tharappel JC , Glauert HP , Spear BT , Robertson LW , Appel KE , Buchmann A 2006 PCB 153, a non-dioxin-like tumor promoter selects for β-catenin (Catnb)-mutated mouse liver tumors Toxicol Sci 93 34 - 40
Theodorakis CW , D’surney SJ , Bickham JW , Lyne TB , Bradley BP , Hawkins WE , Farkas WL , McCarthy JF , Shugart LR 1992 Sequential expression of biomarkers in bluegill sunfish exposed to contaminated sediment Ecotoxicology 1 45 - 73
Theodorakis CW , Shugart LR 1997 Genetic ecotoxicology II: population genetic structure in radionuclide-contaminated mosquitofish (Gambusia affinis) Ecotoxicology 6 335 - 354
Turuspekov Y , Adams RP , Kearney CM 2002 Genetic diversity in three perennial grasses from Semipalatinsk nuclear testing region of Kazakhstan after long-term radiation exposure Biochem Syst Ecol 30 809 - 817
Ungerer J , Thomas P 1996 Transport and accumulation of organochlorines in the ovaries of Atlantic croaker (Micropogonias undulatus) Mar Environ Res 42 167 - 171
Von Westernhagen H , Hoar WS , Randall DJ 1988 Sublethal effects of pollutants on fish eggs and larvae;Fish physiology. Volume XIA Academic Press Inc San Diego, USA 253 - 346
Walker CH , Walker C , Livingstone DR 1994 The ecotoxicology of persistent pollutants in marine fish-eating birds;Persistent pollutants in marine ecosystems Pergamon Press Oxford, USA 211 - 232
Weis JS , Weis P 1989 Effects of environmental pollutants on early fish development Rev Aquat Sci 1 45 - 73
Whysner J , Montandon F , McClain RM , Downing J , Verna LK , Steward RE , Williams GM 1998 Absence of DNA adduct formation by phenobarbital, polychlorinated biphenyls, and chlordane in mouse liver using the 32P-postlabeling assay Toxocol Appl Pharmacol 148 14 - 23
Xia C , Lam JCW , Wu X , Xie Z , Lam PKS 2012 Polychlorinated biphenyls (PCBs) in marine fishes from China: Levels, distribution and risk assessment Chemosphere 89 944 - 949