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DNA Damage and Effects on Glutathione-S-Transferase Activity Induced by Atrazine Exposure in Zebra?sh(Danio rerio)

Lusheng Zhu,Xiaoli Dong,Hui Xie,Jun Wang,Jinhua Wang,Jun Su,Changwei Yu College of Resources and Environment,Shandong Agriculture University,Taian271018,China

Received14December2009;revised19January2010;accepted26January2010

ABSTRACT:This study was undertaken to investigate the protective effect of atrazine(2-chloro-4-(ethyl-amino)-6-(isopropylamino)-S-triazine)on the activity of glutathione-S-transferase(GST)and DNA damage in males and females of adult zebra?sh(Danio rerio).Zebra?sh were exposed to control and three treat-ments(0.01,0.1,and1mg/L)of atrazine for5,10,15,20,and25days.The results indicated that,for males,the GST activity at lower atrazine concentrations(0.01and0.1mg/L)was markedly higher than that of the controls throughout the duration of the experiment while there was a signi?cant inhibition of the GST activity at1mg/L atrazine at days5and20.For females,a signi?cant increase was detected at0.1 mg/L on the days5and15and at0.01mg/L on day20.The DNA damage in zebra?sh was evaluated using the comet assay;the olive tail moments obtained for hepatopancreas were enhanced after treat-ment with different concentrations of atrazine on days5,10,15,20,and25.The DNA damage increased with increasing atrazine concentrations,indicating that genotoxicity of atrazine and signi?cant differences was found compared to the controls.In conclusion,these?ndings provide further evidence of the effects of atrazine on aquatic ecosystems.#2010Wiley Periodicals,Inc.Environ Toxicol00:000–000,2010.

Keywords:atrazine;zebra?sh;toxicity;glutathione-S-transferase;comet assay

INTRODUCTION

In the recent years,there has been growing concern over the widespread use of herbicides that contaminate the aquatic environment.Herbicides are released into aquatic ecosystems as a consequence of agriculture runoff,rains, irrigation waters,wetland applications,etc.Among the various herbicides used,atrazine(2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine)attracts special attention because of its high stability and persistence in the aquatic environment.

Atrazine,which was introduced in the1950s,is a selec-tive pre-and early-postemergence herbicide extensively used in agriculture to suppress the growth of weeds.It is considered a moderately persistent chemical in the environ-ment with a half-life ranging from a few days to months (Khan and Saidak,1981;Jones et al.,1982)and is most fre-quently detected in aquatic ecosystem(Dubus et al.,2000; Konstantinou et al.,2006).According to the EPA(1991), the maximum concentration level for atrazine in water should be3l g/L,but atrazine has been detected in ground-water at levels up to65l g/L.In aquatic ecosystems,the ecotoxicological relevance of atrazine has been widely studied using various marine plants and marine animals, including?sh(Huber,1993;Solomon et al.,1996;Spano et al.,2004).In the recent times,?sh are increasingly being used as biomarkers for the evaluation of the health of aquatic ecosystems,which may help identify possible

Correspondence to:L.S.Zhu;e-mail:lushzhu@https://www.sodocs.net/doc/8d7527126.html,

Contract grant sponsor:National Natural Science Foundation of China.

Contract grant numbers:20477022,40801203.

Contract grant sponsor:National Key Technology R&D Program.

Contract grant number:2008BADA4B05.

Contract grant sponsor:Postdoctoral Science Foundation of China.

Contract grant number:20080431215.

Published online in Wiley InterScience(https://www.sodocs.net/doc/8d7527126.html,).

DOI10.1002/tox.20575

C2010Wiley Periodicals,Inc.

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environmental problems(Bombail et al.,2001).The nega-tive effects of atrazine on?sh have been evaluated in terms of several parameters,including biochemical(Elia et al., 2002),histological(Nesˇkovic et al.,1993),behavioral (Steinberg et al.,1995),and growth and reproduction(Hus-sein et al.,1996;Wiegand et al.,2001;Alvarez and Fuiman, 2005).Although acute poisoning of?sh by atrazine is highly unlikely,because this chemical is present in low concentrations in water,the possible chronic toxic effects of atrazine pose a health risk.

Atrazine is metabolized by a complex metabolic process involving several groups of xenobiotic metabolizing enzymes such as cytochrome P450isozymes and glutathi-one-S-transferase(GST).GST is a phase II enzyme with a determinant function in the detoxi?cation processes(Thom et al.,2001);it catalyzes the conjugation of several xenobi-otics with glutathione(GSH).GST is also important for antioxidative protection and xenobiotic metabolism and for the detoxi?cation of reactive oxygen species(ROS)in cells (Edwards et al.,2000).Thus,the induction of GST is con-sidered bene?cial to handle environmental stress(Van der Oost et al.,2003)and has been considered as a useful bio-marker for monitoring environmental pollution in a variety of marine organisms,including invertebrates,mussels,and ?sh(Bozcaarmutlu et al.,2009).Currently,even though GST activity has been investigated in several?sh species (Wiegand et al.,2000,2001;Schmidt et al.,2005;Mart?′nez-Go′mez et al.,2006;Donham et al.,2007;Wu et al.,2007; Carletti et al.,2008;Chang et al.,2008;Gallagher et al., 2008;Pesce et al.,2008;Ballesteros et al.,2009;Salaberria et al.,2009),there is a lack of information on the effect of atrazine on the GST enzyme in adult zebra?sh(Danio rerio),and further investigations on?sh are required, because they are expected to serve as water pollution biomarkers.

Apart from using enzymatic biomarkers,other parame-ters,such as DNA damage,should be evaluated for assess-ing the consequences of exposure to and the metabolism of herbicides(Mitchelmore and Chipman,1998).According to some authors,the DNA damage induced by atrazine exposure could be evaluated by using the comet assay(Zel-jezic et al.,2006;Singh et al.,2008).The comet assay [alkaline single-cell gel electrophoresis(SCGE)]is a widely used technique to detect DNA damage due to envi-ronmental stress and has been used for the analysis of bio-markers of environmental pollution(Amado et al.,2006). Jarvis and Knowles(2003)determined the sensitivity of the alkaline comet assay for the detection of strand breaks in the DNA of zebra?sh larvae and also used the comet assay to assess the toxic effects on zebra?sh embryos(Sun et al., 2004;Kosmehl et al.,2008).Diekmann et al.(2004b)stud-ied the genotoxic effects in zebra?sh exposed to4-nitroqui-noline-1-oxide in a complete life-cycle test by using the comet assay.Additionally,the genotoxicity of different concentrations of atrazine was assessed using the comet assay,with Cichlidae(Oreochromis niloticus)as the test system(de Campos Ventura et al.,2008).However,to the extent of our knowledge,reports on genotoxicity of atrazine in adult zebra?sh are scarce.

In previous studies performed at our laboratory,we examined the effect of atrazine exposure on antioxidative enzymes and DNA damage in earthworm(Eisenia foetida) and horsebean(Vicia faba)and found that atrazine at con-centrations of2.5,5,and10mg/kg promotes both the activ-ity of antioxidative enzymes and DNA damage(Song et al., 2009a,b);additionally,the cytochromes P450responses of zebra?sh exposed to atrazine were also studied in our labo-ratory recently(Dong et al.,2009).The aim of this study was to assess the effects of atrazine on GST activity and genotoxicity at lower doses(0,0.01,0.1,and1mg/L)in zebra?sh.

MATERIALS AND METHODS

Chemicals

Atrazine of99.9%purity was purchased from AccuStan-dard(New Haven,CT).All other chemicals used to assess the enzymatic activity were of analytical purity and were obtained from Sigma Chemical Co and Shanghai Sangon Biological Engineering Technology and Service Co.

Fish Treatment and Toxicity Test

Adult male(mean body weight,0.2760.01g;length,2.47 60.03cm)and female(mean body weight,0.3360.01g; length,2.4860.02cm)zebra?sh(Danio rerio)used in this study were bought from an aquarium specializing in tropi-cal?sh species.The?sh were acclimated in the laboratory for2weeks before the commencement of the experiments to ensure that they were disease-free.

Males and females were housed separately;they were both exposed to the control and three different concentra-tions of atrazine(0.01,0.1,and1mg/L)in dechlorinated tap water under constant day/night rhythm in large40-L glass aquaria provided with a?lter and continuous aeration. About100zebra?sh for each sex were allocated per tank. During the experimental period,light regiment(12h:12h), temperature(268C618C),and other conditions(e.g.,oxy-gen saturation and pH)were in a range appropriate for maintenance and breeding of zebra?sh(Diekmann et al., 2004a);?sh were fed ad libitum for5min twice a day with commercial?sh pellets and starved24h before the test to avoid prandial effects and to prevent deposition of feces in the course of the assay;the remainder food was removed after feeding.Furthermore,50%of the contaminated water was changed every2days for the duration of the exposure period and sampled at days5,10,15,20,and25.

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Environmental Toxicology DOI10.1002/tox

Preparation of Hepatopancreas Microsomes

All procedures were performed at48C using cold buffers and centrifuges to prevent breakdown of enzymes.

Hepatopancreas were promptly removed from the?sh under ice-cold conditions and rinsed with ice-cold0.15M KCl to completely eliminate blood traces.Subsequently, the hepatopancreas were homogenized in seven volumes of ice-cold homogenization buffer[0.1M sodium phosphate buffer,pH7.5,containing1mM ethylene diamine tetraace-tic acid(EDTA),0.1mM dithiothreitol(DTT),and0.1mM phenylmethylsulfonyl?uoride(PMSF)]supplemented with 10%(v/v)glycerol using a glass-Te?on homogenizer.Sub-sequently,the homogenate obtained was?rst centrifuged at13,0003g for30min at48C,and the supernatant was centrifuged at105,0003g for1h at48C.The microsomal pellet was resuspended in homogenization buffer supple-mented with20%(v/v)glycerol for future use.

Enzyme Assays

The activity of microsomal GST was determined by using a spectrophotometer(Varian Cary50,USA),according to the methods described by Habig et al.(1974),with1-chloro-2,4-dinitrobenzene(CDNB)as a model substrate.Hepato-pancreas of three?sh were assayed,with three replicates per sample.An extinction coef?cient of9.6mM21cm21 was used for CDNB,and changes in absorbance at340nm were recorded for3min.Results were expressed as nano-mole per minute per milligram of protein.Total protein concentrations were determined using the method of Brad-ford(1976),with bovine serum albumin as a standard. DNA Damage in Zebra?sh Hepatopancreas

The following pretreatment process is mostly done accord-ing to Diekmann et al.(2004b).Three?sh of each sex were collected on the days5,10,15,20,and25,following the application of atrazine,with three replicates per sample.

Zebra?sh were anesthetized with benzocaine,opened ventrally,and the blood was removed by perfusion with PBS.Hepatopancreas were dissected,rinsed in PBS,sup-plemented with gentamycin(50l g/mL),streptomycin–pen-icillin solution(20l L/mL),and amphotericin B(2.5l g/ mL),and incubated in0.05%trypsine in PBS with0.02% EDTA for15min.After?ltration through a70-l m nylon cloth into a stopping solution containing PBS with10% fetal bovine serum(FBS),cells were centrifuged at1803g for10min and incubated with erythrocyte lysis buffer(144 mM NH4Cl,17mM Tris,and pH7.2).Lysis was stopped with medium M199(Hank’s modi?cation,20mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,350mg/L NaHCO3,10%FBS,1%streptomycin–penicillin),and, after another centrifugation,pellets were resuspended in sterile medium M199.The resulting cell suspensions were processed in the comet assay.Cell viability was determined using a1%solution of Trypan Blue in PBS.

The comet assay was performed according to Singh et al.(1988),with slight modi?cations.The cell suspension was mixed with100l L of0.7%low-melting agar(LMA) in PBS at378C and pipetted onto fully frosted slides pre-coated with a layer of100l L0.8%normal melting agar. After solidi?cation on ice,another layer of85l L LMA was added,and the slides were immersed into a lysis solu-tion(2.5M NaCl,10mM Tris,100mM Na2EDTA(pH 10.0),1%Na-sarcosinate,10%dimethyl sulfoxide,and1% Triton X-100).Slides were then incubated in an electropho-resis tank containing300mM NaOH with1mM Na2EDTA for20min before electrophoresis for15min at25V(300 mA).The slides were then neutralized(0.4M Tris and pH 7.5)thrice at5-min intervals and stained with40-l L ethi-dium bromide(13l g/mL)for?uorescence microscopy analysis(Olympus BX71?uorescence microscope)using a digital imaging system.The images of the SCGE were ana-lyzed last using Comet Assay Software Project to measure various comet parameters(Konca et al.,2003).One hun-dred cell cores on each slide were counted.The parameters used to quantify the extent of DNA damage was the olive tail moment(OTM).OTM is the product of the distance between the center of gravity of the head and the center of gravity of the tail and percent tail DNA.

Statistics

The data in this study were analyzed with the statistical package for social sciences(SPSS)program(Standard Ver-sion11.5,SPSS).Least signi?cance difference(LSD)test was used to determine the differences between treatments and control.Analysis of variance was used to determine the differences between duration and concentrations.The prob-ability level used for the statistical signi?cance was P\ 0.01.All the values were presented as mean6standard deviation(SD).

RESULTS

Effect of Atrazine on GST Activity in

Zebra?sh Hepatopancreas

As shown in Figure1,in the case of males,GST activity was notably higher than that in controls at most of atrazine concentrations at all time points,except for the1mg/L treatment on days5and20,when it was markedly lower. For females,the GST activity on day5when treated with 0.01and0.1mg/L of atrazine was higher and that at1mg/ L lower than that of the controls;the difference between the activity levels was statistically signi?cant at0.1mg/L of atrazine.GST activity at days10,15,and20was higher than that of the controls at concentrations of0.01,0.1,and

3 DNA DAMAGE AND EFFECTS ON GST ACTIVITY

Environmental Toxicology DOI10.1002/tox

1mg/L,and the differences between the activity levels were statistically signi?cant at days 15and 20of 0.1and 0.01mg/L atrazine treatment,respectively.At day 25,GST activity decreased slightly but not signi?cantly after expo-sure to atrazine at concentrations of 0.01,0.1,and 1mg/L.

DNA Damage Induced by Atrazine

Following exposure,cell viability was consistently [85%in all treatments,allowing the conduction of the comet assay.

In the comet assay,the broken strands of DNA migrate toward the anode when an electric ?eld is applied,creating an image that resembles a comet.The amount of DNA that migrates away from the nuclei is used to assess the extent of DNA damage.The DNA migration in hepatopancreas samples increased with an increase in the concentration of atrazine (Fig.2).In comparison,the images obtained for the control cells demonstrated a nucleoid core with zero or minimal DNA migrating to the tail region [Fig.2(a)].In comet assay,slight DNA migration was found under the control and atrazine exposure increased the DNA migration signi?cantly.Upon treatment with 0.01,0.1,and 1mg/L of atrazine,DNA breakage was induced,and most of the DNA migrated away from the nuclei [Fig.2(b,c,d)].

Figure 3summarizes the results for all concentrations of atrazine,in terms of the OTM for zebra?https://www.sodocs.net/doc/8d7527126.html,pared to the other parameters of comet assay,OTM is more sensitive to differences in the gel and subsequent variability in DNA migration and may not demonstrate a linear dose response (Mitchelmore and Chipman,1998;Mitchelmore et al.,1998).As shown in Figure 3,an enhancement of the effects of DNA damage in zebra?sh hepatopancreas was identi?ed in SCGE analysis,with average OTMs following atrazine treatment.Signi?cant effects were induced by

increasing

Fig.1.Effect of atrazine on the GST activity of zebra?sh.Pro is the abbreviation of protein.Each point is the mean of three replicates.Error bars represent standard deviation (SD).*,Symbol indicates signi?cant differences at P \0.01level,respectively,compared to the control.Different letters above columns indicate signi?cant differences at P \0.01level between

treatments.

Fig.2.(a)Control zebra?sh liver cells with subsequent analysis by the comet assay.Nuclei consist of a head (nucleiod core)with no or minimal DNA migrating into the region.(b)Nuclei from zebra?sh liver cells exposed to atrazine of 0.01mg/L consist of a head (nucleoid core)with DNA migrating into the tail region as a result of strand breakage.(c)Zebra?sh liver cell as following exposure to 0.1mg/L atrazine with subsequent analysis by the comet assay.(d)The majority of zebra?sh liver cell nucleoid DNA has migrated into the tail region as a result of extensive strand breakage produced by atrazine of 1mg/L.

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Environmental Toxicology DOI 10.1002/tox

the doses of atrazine ranging from 0.01to 1mg/L on days 5,10,15,20,and 25(P \0.01).DNA fragmentation,measured as OTM,showed a dose-response curve,indicat-ing that DNA damage increased with an increase in the atrazine concentration.

DISCUSSION

Many enzymatic activities and low-molecular weight pro-teins,such as GSH of the phase II biotransformation sys-tem,have been considered as biomarkers of environmental pollution (Saint-Denis et al.,1999).According to some authors,the GST-mediated conjugation of atrazine to GSH is one of the most important pathways of atrazine metabo-lism in animals,and oxidative stress arises as side effect (Egaas et al.,1993;Elia et al.,2002).In our study,GST was used as a biomarker for the evaluation of the effect of atrazine on zebra?sh.

Besides functioning as an enzyme of phase II biotrans-formation,GST is also capable of detoxifying ROS as a result of a direct antioxidant action,and its activity in ?sh cells subjected to oxidative stress (Wu et al.,2007;Mon-teiro et al.,2009;Oliveira et al.,2009).Therefore,it is a potential biomarker not only for pollutants that are detoxi-?ed by GST but also for the very large number of pollutants capable of generating oxidative stress.As noted by Pereira et al.(2009),oxidative damage can occur when antioxidant and detoxifying systems are de?cient and unable to neutral-ize the active intermediates produced by toxins and their metabolites.Our study presents evidence that the GST activity is different for male and female zebra?sh.For males,atrazine treatment induced overall stimulation of the GST activity,except at 1mg/L atrazine on the days 5and 20when it induced a signi?cant inhibition.For females,atrazine exposure at all concentrations increased the overall GST activity from day 5to day 20,except at 1mg/L on day 5and decreased the overall GST activity on day 25at all concentrations of atrazine;signi?cant stimulation was detected at a concentration of 0.1mg/L on days 5and 15and at 0.01mg/L on day 20.Because GST is involved in both detoxi?cation and lipid peroxidation (LPO)processes,it can be hypothesized that the increased GST activity in the hepatopancreas after exposure to atrazine could appear as a result of LPO induction in hepatic tissue,which is easily susceptible to ROS with higher polyunsaturated fatty acid contents (Nimptsch et al.,2005).It also indicates that this enzyme was induced either by the detoxi?cation of hydroper-oxides or by the GSH conjugation as part of phase II biotrans-formation of xenobiotics (Thomaz et al.,2009).The increase in the GST activity observed in this study is consistent with the ?ndings of Contardo-Jara and Wiegand (2008)who treated specimens of black worm (Lumbriculus variegates )with atrazine and Ballesteros et al.(2009)who treated brain samples of Cyprinodontiformes (Jenynsia multidentata )with endosulfan.Likewise,inductions of GST activity have also been demonstrated to be induced in zebra?sh embryos by atrazine treatment (Wiegand et al.,2001).On the contrary,no signi?cant alteration in GST activity was observed in adult zebra?sh exposed to d -endotoxin or cyclophosphamide (Gri-solia et al.,2009)and in liver of common carp exposed to ala-chlor (Mikula et al.,2009).Rakotondravelo et al.(2006)reported that the levels of GST did not increase signi?cantly in atrazine-exposed aquatic midge Chironomidae (Chironomus tentans ).These obviously contradictory results probably owing to a different answer of the enzyme at different xeno-biotics concentrations or may also due to the individual dif-ferences between species.Additionally,the decrease of GST activity at high concentrations seems to be connected with its inactivation by ROS (Lushchak et al.,2009);how-ever,another correlative factor is the possibility of the exhaustion of GSH (Elumalai et al.,2007;Yi et al.,2007).Additionally,a gender difference was noted between male and female zebra?sh in the level of GST

activity:

Fig.3.Effect of atrazine on zebra?sh liver comet olive tails moment.Each point is the mean of three replicates.Error bars represent standard deviation (SD).*Symbol indicates signi?cant differences at P \0.01level,respectively,com-pared to the control.Different letters above columns indicate signi?cant differences at P \0.01level between treatments.

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Environmental Toxicology DOI 10.1002/tox

males exhibited a higher level of activity than females. Similarly,in other species such as crab(Carcinus maenas) and bulltrout(Notesthus robusta),GST activity was found to be higher in males than in females(Ruus et al.,2002; Pereira et al.,2009).Moreover,female brown bullheads (Amerius nebulosus)of Lake Apopka,Florida(well known for its organochlorine contamination),demonstrated lower levels of hepatic GST conjugation activity and expression when compared with those from a minimally polluted lake (Lake Woodruff)(Gallagher et al.,2001).Conversely, Chang et al.(2008)reported that there was no clear differ-ence between male and female striped bass with regard to hepatic GST activity.However,furthermore,more studies are still needed to explore the relative exact mechanism on gender differences on GST activity.

Environmental contaminants may result in membrane LPO and ultimate DNA damage,directly by the action of parental compounds or their metabolites or indirectly by the generated ROS(Oliveira et al.,2009).In addition, according to Chakraborty et al.(2009),the signi?cant induction of GST activity could protect against DNA-dam-age in animals;they attributed this effect to the possible detoxi?cation via conjugation with GSH.Therefore,apart from using GST enzymes as biomarkers,we also use the DNA damage index to evaluate atrazine toxicity.In this study,the enhancement of DNA damage was due to oxida-tive stress,indicating that ROS accumulation in tissues caused subsequent DNA damage or due to the activation of DNA repair mechanisms induced by atrazine in zebra?sh hepatopancreas.The accumulation of ROS may be impli-cated in the reduction in the detoxifying capabilities of GST.The comet assay,which is widely used to evaluate the genotoxicity of chemicals in the environment,was used to assess the toxic effects on zebra?sh embryos and liver (Deventer,1996;Sun et al.,2004;Kosmehl et al.,2008; Seitz et al.,2008).Our results indicated that the DNA dam-age in zebra?sh hepatopancreas increased with increasing atrazine concentration,indicating that atrazine-induced DNA damage in zebra?sh and the levels of DNA damage imposed by atrazine could be quanti?ed by comet assay. Our studies with zebra?sh could con?rm the genotoxicity of atrazine,which was evaluated by the evidence of frag-mentation of the genetic material of different organisms exposed to this herbicide(Clements et al.,1997;Tennant et al.,2001;Garaj-Vrhovac and Zeljezic,2002;Ribas et al., 1995).The toxicological effect of atrazine originates from its oxidizing action;once inside the cell,in the presence of cellular reductants,it induces DNA fragmentation or break-age of DNA strands,which is considered to be premuta-genic change(Kammann et al.,2001).Our results with zebra?sh could also con?rm that the comet assay using hep-atopancreas of?sh seemed to offer the same ef?ciency for the detection of the genotoxicity of chemicals as that obtained using erythrocytes of?sh,which was used by some authors(Belpaeme et al.,1996;Nacci et al.,1996;Mitchelmore and Chipman,1998;Ateeq et al.,2005;Degu-chi et al.,2007).

Interestingly,GST activity exhibited a signi?cant rise at most of atrazine treatments,whereas signi?cant increases in DNA damage were detected by alkaline SCGE assay. The lack of correlation between these biomarkers seems to indicate that the impacts of the pollutants to organisms could express in different levels,which start from the bio-moleculars and then biological reactions of the enzymes followed.Zebra cells seem able to repair minor DNA dam-age made by this chemical administered at environmental doses.Additionally,the particular behavior is in agreement with the?ndings obtained by Binelli et al.(2009),who also reported that the controversial results could shed some light in scienti?c literature about the cyto-and genotoxicity of the chemicals.

CONCLUSIONS

To evaluate atrazine ecotoxicology in water,the effect of atrazine on the activity of GST and DNA damage was investigated in zebra?sh(Danio rerio)hepatopancreas.The analysis of the differences between treatments reveals that atrazine induces GST activity and DNA damage on zebra-?sh,which may be important mechanisms underlying the toxic effects of this chemical on zebra?sh;however,we cannot clarify whether these effects were the exclusive mechanisms for atrazine induction.To con?rm this,other methods of testing using other species may be required.In conclusion,these?ndings provide a scienti?c basis for the rational evaluation of atrazine ecotoxicology. REFERENCES

Alvarez MD,Fuiman LA.2005.Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae.Aquat Toxicol74:229–241.

Amado LL,Robaldo RB,Geracitano L,Monserrat JM,Bianchini A.2006.Biomarkers of exposure and effect in the Brazilian ?ounder Paralichthys orbignyanus(Teleostei:Paralichthyidae) from the Patos Lagoon estuary(Southern Brazil).Marine Pollut Bull52:207–213.

Ateeq B,Farah MA,Ahmad W.2005.Detection of DNA damage by alkaline single cell gel electrophoresis in2,4-dichlorophe-noxyacetic-acid-and butachlor-exposed erythrocytes of Clarias batrachus.Ecotoxicol Environ Safety62:348–354. Ballesteros ML,Wunderlin DA,Bistoni MA.2009.Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan.Ecotoxicol Environ Safety72:199–205. Belpaeme K,Deldeke K,Zhu L,Kirsch-Volders M.1996.Cytoge-netic studies of PCB77on brown trout(Salmo trutta fario) using the micronucleus test and the alkaline comet assay.Muta-genesis11:485–492.

Binelli A,Parolini M,Cogni D,Pedriali A,Provini A.2009.A multi-biomarker assessment of the impact of the antibacterial

6ZHU ET AL.

Environmental Toxicology DOI10.1002/tox

trimethoprim on the non-target organism Zebra mussel(Dreis-sena polymorpha).Comp Biochem Physiol C150:329–336. Bombail V,Dennis AW,Gordon E,Batty J.2001.Application of the comet and micronucleus assays to butter?sh(Pholis gunnel-lus)erythrocytes from the Firth of Forth,Scotland.Chemo-sphere44:383–392.

Bozcaarmutlu A,Sapmaz C,Aygun Z,Arinc?E.2009.Assessment of pollution in the west black sea coast of turkey using bio-marker responses in?sh.Marine Environ Res67:167–176. Bradford MM.1976.A rapid sensitive method for the quanti?ca-tion of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem72:248–254.

Carletti E,Sulpizio M,Bucciarelli T,Boccio PD,Federici L,DiI-lio C.2008.Glutathione S-transferases from Anguilla anguilla liver:Identi?cation,cloning and functional characterization. Aquat Toxicol90:48–57.

Chakraborty A,Ferk F,Simic T,Brantner A,Dusˇinska′M,Kundi M,Hoelzl C,Nersesyan A,Knasmu¨ller S.2009.DNA-protec-tive effects of sumach(Rhus coriaria L.),a common spice: Results of human and animal studies.Mutat Res661:10–17. Chang S,Donham RT,Luna AD,Morin D,Jewell WT,Tjeerdema RS.2008.Characterization of cytosolic glutathione S-transfer-ases in striped bass(Morone saxitilis).Ecotoxicol Environ Safety69:58–63.

Clements C,Ralph S,Petras M.1997.Genotoxicity of select her-bicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis(comet)assay.Environ Mol Mut 29:277–288.

Contardo-Jara V,Wiegand C.2008.Biotransformation and antiox-idant enzymes of Lumbriculus variegates as biomarkers of con-taminated sediment exposure.Chemosphere70:1879–1888.

de Campos Ventura B,de Fransceschi de Angelis D,Marin-Morales MA.2008.Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus(Perciformes. Cichlidae)detected by the micronuclei test and the comet assay. Pest Biochem Physiol90:42–51.

Deguchi Y,Toyoizumi T,Masuda S,Yasuhara A,Mohri S, Yamada M,Inoue Y,Kinae N.2007.Evaluation of mutagenic activities of leachates in land?ll sites by micronucleus test and comet assay using gold?sh.Mutat Res627:178–185. Deventer K.1996.Detection of genotoxic effects on cells of liver and gills of B.rerio by means of single cell gel electrophoresis. Bull Environ Contam Toxicol56:911–918.

Diekmann M,Hultsch V,Nagel R.2004a.On the relevance of genotoxicity for Fish populations.I.Effects of a model genotox-icant on Zebra?sh(Danio rerio)in a complete life-cycle test. Aquat Toxicol68:13–26.

Diekmann M,Waldmann P,Schnurstein A,Grummt T,Braunbeck T,Nagel R.2004b.On the relevance of genotoxicity for?sh populations.II.Genotoxic effects in zebra?sh(Danio rerio) exposed to4-nitroquinoline-1-oxide in a complete life-cycle test.Aquat Toxicol68:27–37.

Dong XL,Zhu LS,Wang JH,Wang J,Xie H,Hou XX,Jia WT. 2009.Effects of atrazine on cytochrome P450enzymes of zebra?sh(Danio rerio).Chemosphere77:404–412.

Donham RT,Luna AD,Chang S,Morin D,Jewell WT,Tjeerdem RS.2007.Characterization of cytosolic glutathione S-transfer-

ases in California Halibut(Paralichthys californicus).Ecotoxi-col Environ Safety66:133–138.

Dubus IG,Hollis JM,Brown CD.2000.Pesticides in rainfall in Europe.Environ Pollut110:331–344.

Edwards R,Dixon DP,Walbot V.2000.Plant glutathione S-trans-ferases:Enzymes with multiple functions in sickness and in health.Trends Plant Sci5:193–198.

Egaas E,Skaare JU,Svendsen NO,Sandvik M,Falls JG,Dauter-man WC,Collier TK,Netland J.1993.A comparativestudy of effects of atrazine on xenobiotic-metabolizing enzymes in?sh and insect,and of the in-vitro phase-II atrazine metabolism in some?sh,insects,mammals and one https://www.sodocs.net/doc/8d7527126.html,p Bio-chem Phys C106:141–149.

Elia AC,Waller WT,Norton SJ.2002.Biochemical responses of Bluegill Sun?sh(Lepomis macrochirus.Ra?nesque)to atrazine induced oxidative stress.Bull Environ Contam Toxicol68:809–816.

Elumalai M,Antunes C,Guilhermino L.2007.Enzymatic bio-markers in the crab Carcinus maenas from the Minho River es-tuary(NW Portugal)exposed to zinc and mercury.Chemo-sphere66:1249–1255.

Gallagher EP,Gross TS,Sheehy KM.2001.Decreased glutathi-one S-transferase expression and activity and altered sex ste-roids in Lake Apopka brown bullheads(Ameriurus nebulosus). Aquat Toxicol55:223–227.

Gallagher EP,LaVire HM,Bammler TK,Stapleton PL,Beyer RP, Farin FM.2008.Hepatic expression pro?ling in smolting and adult coho salmon(Onchorhynchus kisutch).Environ Res 106:365–378.

Garaj-Vrhovac V,Zeljezic D.2002.Assessment of genoma dam-age in a population of Croatian workers employed in pesticide production by chromosomal aberration analysis,micronucleus assay and Comet assay.J Appl Toxicol22:249–255.

Grisolia CK,Oliveira R,Domingues I,Oliveira-Filho EC,Mon-erat RG,Soares AMVM.2009.Genotoxic evaluation of differ-ent d-endotoxins from Bacillus thuringiensis on zebra?sh adults and development in early life stages.Mutat Res672:119–123. Habig W,Pabst MJ,Jacoby WB.1974.Glutathione-S-transferase: The?rst step in mercapturic acid formation.J Biol Chem 249:1730–1739.

Huber W.1993.Ecotoxicological relevance of atrazine in aquatic systems.Environ Toxicol Chem12:1865–1881.

Hussein SY,El-Nasser MA,Ahmed https://www.sodocs.net/doc/8d7527126.html,parative stud-ies on the effects of herbicide atrazine on freshwater?sh Ore-chromis nilotilus and Chrysichteres auratus at Assiut,Egypt. Bull Environ Contam Toxicol57:503–510.

Jarvis RB,Knowles JF.2003.DNA damage in zebra?sh larvae induced by exposure to low-dose rate c-radiation:Detection by the alkaline comet assay.Mutat Res541:63–69.

Jones TW,Kemp WM,Stevenson JC,Means JC.1982.Degrada-tion of atrazine in estuary water,sediments and soils.J Environ Microbiol45:97–102.

Kammann U,Bunke M,Steinhart H,Theobald N.2001.A perma-nent?sh cell line(EPC)for genotoxicity testing of marine sedi-ments with the comet assay.Mutat Res498:61–77.

Khan SU,Saidak WJ.1981.Residues of atrazine and its metabo-lites after prolonged usage.Weed Res21:9–12.

7 DNA DAMAGE AND EFFECTS ON GST ACTIVITY

Environmental Toxicology DOI10.1002/tox

Konstantinou IK,Hela DG,Albanis TA.2006.The status of pesti-cide pollution in surface waters(rivers and lakes)of Greece, Part1:Review on occurrence and levels.Environ Pollut 141:555–570.

Kon′ca K,Lankoff A,Banasik A,Lisowska H,Kuszewski T, Go′zdz S,Koza Z,Wojcik A.2003.A cross-platform public do-main PC image-analysis program for the comet assay.Mutat Res534:15–20.

Kosmehl T,Hallare AV,Braunbeck T,Hollert H.2008.DNA damage induced by genotoxicants in zebra?sh(Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake(The Philippines)as meas-ured by the comet assay.Mutat Res650:1–14.

Lushchak OV,Kubrak OI,Storey JM,Storey KB,Lushchak VI. 2009.Low toxic herbicide Roundup induces mild oxidative stress in gold?sh tissues.Chemosphere76:932–937.

Mart?′nez-Go′mez C,Campillo JA,Benedicto J,Ferna′ndez B, Valde′s J,Garc?′a I,Sa′nchez F.2006.Monitoring biomarkers in ?sh(Lepidorhombus boscii and Callionymus lyra)from the northern Iberian shelf after the Prestige oil spill.Marine Pollut Bull53:305–314.

Mikula P,Blahova J,Kruzikova K,Havelkova M,Nemethova D, Hulak M,Svobodova Z.2009.Effects of the herbicide LASSO MTX(alachlor42%W/V)on biometric parameters and liver biomarkers in the common carp(Cyprinus carpio).Pest Bio-chem Physiol93:13–17.

Mitchelmore CL,Chipman JK.1998.Detection of DNA strand breaks in brown trout(Salmo trutta)hepatocytes and blood cells using the single cell gel electrophoresis(comet)assay.Aquat Toxicol41:161–182.

Mitchelmore CL,Birmelin C,Livingstone DR,Chipman JK. 1998.Detection of DNA strand breaks in isolated Mussel (Mytilus edulis L.)digestive gland cells using the‘‘Comet" assay.Ecotoxicol Environ Safety41:51–58.

Monteiro DA,Rantin FT,Kalinin AL.2009.The effects of sele-nium on oxidative stress biomarkers in the freshwater characid ?sh matrinxa?,Brycon cephalus(Gu¨nther,1869)exposed to or-ganophosphate insecticide Folisuper600BR1(methyl para-thion).Comp Biochem Physiol C149:40–49.

Nacci DE,Cayula S,Jackmin E.1996.Detection of DNA damage in individual cells from marine organisms using the single cell gel assay.Aquat Toxicol35:197–210.

Nesˇkovic NK,Elezovic’I,Karan V,Poleksic’V,Budimir M. 1993.Acute and sub-acute toxicity of atrazine to carp(Cyprinus carpio L.).Ecotoxicol Enviorn Safety25:173–182.

Nimptsch J,Wunderlin DA,Dollan A,P?ugmacher S.2005.Anti-oxidant and biotransformation enzymes in Myriophyllum qui-tense as biomarkers of heavy metal exposure and eutrophication in Suqu?′a River basin(Co′rdoba,Argentina).Chemosphere 61:147–157.

Oliveira M,Maria VL,Ahmad I,Sera?m A,Bebianno MJ, Pacheco M,Santos MA.2009.Contamination assessment of a coastal lagoon(Ria de Aveiro,Portugal)using defence and damage biochemical indicators in gill of Liza aurata—An inte-grated biomarker approach.Environ Pollut157:959–967. Pereira P,Pablo Hde,Subida MD,Vale C,Pacheco M.2009.Bio-chemical responses of the shore crab(Carcinus maenas)in a

eutrophic and metal-contaminated coastal system(o′bidos lagoon,Portugal).Ecotoxicol Environ Safety72:1471–1480. Pesce SF,Cazenave J,Monferra′n MV,Frede S,Wunderlin DA. 2008.Integrated survey on toxic effects of lindane on neotropi-cal?sh:Corydoras paleatus and Jenynsia multidentata.Environ Pollut156:775–783.

Rakotondravelo ML,Anderson TD,Charlton RE,Zhu KY.2006. Sublethal effects of three pesticides on activities of selected tar-get and detoxi?cation enzymes in the aquatic midge,Chirono-mus tentans(Diptera:Chironomidae).Arch Environ Contam Tox51:360–366.

Ribas G,Frenzili G,Barale R,Marcos R.1995.Herbicide-induced DNA damage in human lymphocytes evaluated by the single-cell gel electrophoresis(SCGE)assay.Mutat Res 344:41–54.

Ruus A,Sandvik M,Ugland KI,Skaare JU.2002.Factors in?uencing activities of biotransformation enzymes,concen-trations and compositional patterns of organochlorine contam-inants in members of a marine food web.Aquat Toxicol 61:73–87.

Saint-Denis M,Narbonne JF,Arnaud C,Thybaud E,Ribera D. 1999.Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated arti?cial soil:Effects of ben-zo(a)pyrene.Soil Biol Biochem31:1837–1846.

Salaberria I,Hansen BH,Asensio V,Olsvik PA,Andersen RA, Jenssen BM.2009.Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout(Oncorhynchus mykiss).Toxicol Appl Pharmacol234:98–106.

Schmidt K,Staaks GBO,P?ugmacher S,Steinberg CEW.2005. Impact of PCB mixture(Aroclor1254)and TBT and a mixture of both on swimming behavior,body growth and enzymatic biotransformation activities(GST)of young carp(Cyprinus carpio).Aquat Toxicol71:49–59.

Seitz N,Bo¨ttcher M,Keiter S,Kosmehl T,Manzb W,Hollert H, Braunbeck T.2008.A novel statistical approach for the evalua-tion of comet assay data.Mutat Res652:38–45.

Singh M,Kaur P,Sandhir R,Kiran R.2008.Protective effects of vitamin E against atrazine-induced genotoxicity in rats.Mutat Res654:145–149.

Singh NP,McCoy MT,Tice RR,Schneider EL.1988.A simple technique for quantitation of low levels of DNA damage in indi-vidual cells.Exp Cell Res175:184–191.

Solomon KR,Baker DB,Richards P,Dixon KR,Klaine SJ, LaPoint TW,Kendall RJ,Giddings JM,Giesy JP,Hall JLW, Weiskopf CP,Williams W.1996.Ecological risk assessment of atrazine in North American surface waters.Environ Toxicol Chem15:31–76.

Song Y,Zhu LS,Wang J,Wang JH,Liu W,Xie H.2009a.DNA damage and effects on antioxidative enzymes in earthworm (Eisenia foetida)induced by atrazine.Soil Biol Biochem 41:905–909.

Song Y,Zhu LS,Xie H,Wang J,Wang JH,Liu W,Dong XL. 2009b.Effects of atrazine on DNA damage and antioxidative enzymes in Vicia faba.Environ Toxicol Chem28:1059–1062. Spano L,Tyler CR,van Aerle R,Devos P,Mandiki SNM,Sil-vestre F,Thome JP,Kestemont P.2004.Effects of atrazine on sex steroid dynamics,plasma vitellogenin concentration and

8ZHU ET AL.

Environmental Toxicology DOI10.1002/tox

gonad development in adult gold?sh(Carassius auratus).Aquat Toxicol66:369–379.

Steinberg CEW,Lorenz R,Spie b er OH.1995.Effect of atrazine on swimming behavior of zebra?sh,Brachydanio rerio.Water Res29:981–985.

Sun LW,Qu MM,Li YQ,Wu YL,Chen YG,Kong ZM,Liu Z. 2004.Toxic effects of aminophenols on aquatic life using the Zebra?sh embryo test and the comet assay.Bull Environ Con-tam Toxicol73:628–634.

Tennant AH,Peng B,Kligerman AD.2001.Genotoxicity studies of there triazine herbicides:In vivo studies using the alkaline single cell gel(SCG)assay.Mutat Res493:1–10.

Thom R,Dixon DP,Edwards R,Cole DL,Lapthorn AJ.2001. The structure of a zeta class glutathione S-transferase from Ara-bidopsis thaliana:Characterisation of a GST with novel active-site tyrosine catabolism.J Mol Biol308:949–962.

Thomaz JM,Martins ND,Monteiro DA,Rantin FT,Kalinin AL. 2009.Cardio-respiratory function and oxidative stress biomarkers in Niletilapia exposed to the organophosphate insecticide trichlor-fon(NEGUVONs).Ecotoxicol Environ Safety72:1413–1424. US Environmental Protection Agency Rules and Regulations. 1991.Fed Regist56:3543–3544.Van der Oost R,Beyer J,Vermeulen NP.2003.Fish bioaccumula-tion and biomarkers in environmental risk assessment:A review.Environ Toxicol Pharmacol13:57–149.

Wiegand C,P?ugmacher S,Giese M,Frank H,Steinberg C.2000. Uptake,toxicity,and effects on detoxication enzymes of atra-zine and tri?uoroacetate in embryos of Zebra?sh.Ecotoxicol Environ Safety45:122–131.

Wiegand C,Krause E,Steinberg C,P?ugmacher S.2001.Toxico-kinetics of atrazine in embryos of the Zebra?sh(Danio rerio). Ecotoxicol Environ Safety49:199–205.

Wu YQ,Wang CG,Wang Y,Zhao Y,Chen YX,Zuo ZH.2007. Antioxidant responses to benzo[a]pyrene,tributyltin and their mixture in the spleen of Sebasticus marmoratu.J Environ Sci 19:1129–1135.

Yi XH,Ding H,Lu YT,Liu HH,Zhang M,Jiang W.2007.Effects of long-term alachlor exposure on hepatic antioxidant defense and detoxifying enzyme activities in crucian carp(Carassius auratus).Chemosphere68:1576–1581.

Zeljezic D,Garaj-Vrhovac V,Perkovic P.2006.Evaluation of DNA damage induced by atrazine and atrazine-based herbicide in human lymphocytes in vitro using a comet and DNA diffu-sion assay.Toxicol In Vitro20:923–935.

9 DNA DAMAGE AND EFFECTS ON GST ACTIVITY

Environmental Toxicology DOI10.1002/tox