Comparative Biochemistry and Physiology, Part C 149 (2009) 581–586

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Comparative Biochemistry and Physiology, Part C

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Effects of cadmium on respiratory burst, intracellular Ca2+ and DNA damage in thewhite shrimp Litopenaeus vannamei

Ming Chang, Wei-Na Wang ⁎, An-Li Wang, Ting-Ting Tian, Peng Wang, Ying Zheng, Yuan LiuKey Laboratory of Ecology and Environmental Science in Guangdong Higher Education, College of Life Science, South China Normal University, Guangzhou 510631, PR China

⁎ Corresponding author. Tel./fax: +86 20 8521 7322.E-mail addresses: wangwn@scnu.edu.cn, weina63@y

1532-0456/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.cbpc.2008.12.011

a b s t r a c t

a r t i c l e i n f o

Article history:

Acute effects of heavy meta Received 10 October 2008Received in revised form 21 December 2008Accepted 31 December 2008Available online 7 January 2009

Keywords:CadmiumDNA damageRespiratory burstIntracellular Ca2+ concentrationBiomarkersLitopenaeus vannamei

l ions on shrimp have been an area of intense study worldwide. However, themolecular mechanism by which cadmium-induced injury occurs remains largely unclear, and methods formitigating toxicity in vivo have rarely been reported. In this study, the changes in respiratory burst andintracellular free calcium in haemocytes of pacific white shrimp, Litopenaeus vannamei, after exposure to Cd2+

(CdCl2) were examined using flow cytometry. Meanwhile, DNA damage and repair in haemocytes andhepatopancreas cells were studied using the comet assay. Respiratory burst generation, intracellular Ca2+

concentration ([Ca2+]i) and DNA damage in haemocytes and hepatopancreas cells all exhibited a dose-dependent increase and a time-dependent change after treatment with Cd2+ compared with controls. Theseresults indicate that Cd can induce oxidative stress and DNA damage in the shrimp L. vannamei. Moreover, theresults also demonstrate that these parameters can be used as sensitive indicators of exposure to thisgenotoxicant.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

Aquatic pollution is a serious and growing problem. The increasingnumber of industrial, agricultural and commercial chemicals in theaquatic environment has had deleterious effects on many organisms.Heavy metal contamination in China, for example, is increasinggreatly, especially in sediments. Cadmium (Cd) is recognized as acarcinogen and its concentration has exceeded the environmentallyacceptable level in several areas, including Jiaozhou Bay (Xu et al.,2005). Recent studies (Chandra and Khuda-Bukhsh, 2004; Companyet al., 2004) have focused mainly on heavy metal toxicity in fish ormussels rather than crustaceans; the latter, however, are usually usedfor assessing heavy metal pollution in seawater.

Oxidative stress is increasingly being studied in marine inverte-brates, which can be used as indicators for monitoring pollution incoastal as well as in more remote environments (Valavanidis et al.,2006). Oxidative stress occurs as a consequence of a disturbance in thebalance between the production of reactive oxygen species (ROS) andthe antioxidant defense mechanisms. Overproduction of ROS may leadto oxidative damage to tissue macromolecules including DNA, proteinsand lipids. Xenobiotic-enhanced oxyradical generation is one possiblemechanism of pollution toxicity (Gómez-Mendikute and Cajaraville,2003). Environmental and dietary toxicants, such as copper sulfate,nitrite, ammonia-N, and higher selenium levels, have been reported toincrease the release of superoxide anions inMacrobrachium rosenbergii,

ahoo.com.cn (W.-N. Wang).

l rights reserved.

M. nipponense and Litopenaeus vannamei (Cheng and Wang, 2001;Wang et al., 2004a, 2005, 2006). DNA damage (DNA strand breakage) isone type of oxidative damage and has been used as a biomarker toassess the genotoxicity of pollutants (e.g. Benzo (a) pyrene, Aroclor1254) to marine organisms (Ching et al., 2001). The comet assay hasalready been used on various aquatic organisms as a promisingmethodfor detecting DNAdamage. Flounder and oysters were tested by Nacci etal. (1996); flatfish were examined by Belpaeme et al. (1998), andGuecheva et al. (2001) found that planarians are suitable forgenotoxicity testing using the comet assay. Moreover, Pavlica et al.(2001) found that the comet assay applied to zebra mussel haemocytesis useful in determining the genotoxicity of the aquatic pollutantpentachlorophenol. They found that this assay can be used in all typesof isolated cells, which makes it an appropriate test for monitoringgenotoxic effects on species in their natural habitats.

Calciumhomeostasismay be affected byheavymetals. Calciumactsas a secondary messenger in a variety of cellular processes in differentorganisms. An increase in cytosolic free calcium in mammalian cellscould be the result of calcium release from intracellular stores or aninflux through channels in the plasma membrane. A subsequentdecrease in calcium is ensured by calciumpumps located in the plasmamembrane and the intracellular membrane (Berridge, 1993). Mostheavy metals induce an alteration in the calcium homeostasis ofmussel haemocytes (Marchi et al., 2004). Oxidative stress to living cellsincreases the intracellular concentration of Ca2+, a process responsiblefor subsequent cell death or injury (Oyama et al., 1999). There isevidence suggesting that calcium concentrations can be related toDNAdamage (Golconda et al., 1993).

582 M. Chang et al. / Comparative Biochemistry and Physiology, Part C 149 (2009) 581–586

Cd has been shown to inhibit the mitochondrial electron transferchain and to induce ROS production (Wang et al., 2004b). Moreover,Cd-promoted oxidative stress leads to various types of oxidativedamage (i.e. lipid peroxidation, DNA damage) in certain marine or-ganisms as a result of the formation of ROS (Company et al., 2004;Chelomin et al., 2005). These observations suggest that enhanced ROSproduction may be at the root of Cd toxicity. The most recent researchon DNA damage induced by Cd was carried out in fish (Chandra andKhuda-Bukhsh, 2004) and bivalves (Emmanouil et al., 2007). Littlework has been conducted on crustaceans, although Hook and Lee(2004) examined Cd-induced DNA damage in the embryos of thegrass shrimp. The purpose of the present study was to determine theeffects of Cd-exposure on respiratory burst generation, [Ca2+]i inhaemocytes and DNA damage in haemocytes and hepatopancreascells of pacific white shrimp, L. vannamei.

2. Materials and methods

2.1. Animal collection and maintenance

Litopenaeus vannamei were obtained from a commercial shrimpfarm in Guangdong Province, China, in January 2008. The animals(averaging 8.00±0.53 cm, 5.31±0.56 g) were kept in 30×20×15 cmglass tanks (seventy shrimps/tank) with seawater filtered at 4‰, pH8.0 and controlled temperature (25±2 °C), with continuous watercirculation. The shrimp were fed twice daily with commercial shrimpfood (40% protein, 5.0% fat, 5.0% fiber and 16% ash, designed for pacificwhite shrimp, L. vannamei by Haiyi Products, China) and feeding wasstopped 24 h before treatment. Shrimp were used in the experimentafter acclimation for more than a week.

2.2. Treatment and experimental design

Acute (24 h) cadmium toxicity tests were conducted according torecommendations given by the United States Environmental ProtectionAgency (EPA, 2000), but with slight modifications. Test solutions wereprepared, immediately prior to use, with culture media from stocksmade with CdCl2·2.5H2O (analytical grade, Sigma-Aldrich Chemical, St.Louis, MO, USA) dissolved in deionized water. Test concentrations ofCdCl2·2.5H2O ranged from 8.76×10−2 to 8.76 µmol L−1. Toxicity testsemployed a completely random design consisting of five metaltreatments and a control group; two-fold serial dilutions were used tovary the cadmium concentration. Ten shrimps were randomly selectedand placed in a 50×35×30 cm plastic aquarium exposure chambercontaining 52 L of test solution. Two replicate exposure chambers wereemployed per treatment or control group. Shrimps were not fed duringthe tests. The number of dead individuals in each container wasdetermined after 24-h exposure. An individual was considered to bedead if it was unresponsive to gentle prodding with a pipette tip. Thetests were repeated and the combined results were used to determinelethal concentrations (LC x values, where x is the percentage mortality)estimated from the probit transformed concentration–response curves.

For the assessment of Cd-induced oxidative stress, we examinedthree Cd-exposure levels (0, 4.25 and 8.50 µmol L−1 Cd2+), eachreplicated three times. Approximately 50 shrimp were used in eachreplicate; they were placed in plastic aquaria (50×35×30 cm, watervolume, 52 L) with 4‰ filtered saltwater (pH 8.0); a fairly constanttemperature was maintained (25±2 °C) and the water was aeratedcontinuously using an air stone. The test solutions were renewed daily,in accordancewith the static renewalmethod for toxicity tests (Buikemaet al., 1982). These shrimps were used for the subsequent assays.

2.3. Haemolymph samples

In the Cd-exposure experiments, the haemolymph of nine shrimpfrom each group (0, 4.25, 8.50 µmol L−1 Cd2+) was collected after 0, 6,

12, 24 and 36 h of exposure. Haemolymph was drawn directly fromthe heart of shrimps using sterile syringes with an anticoagulant. Thehaemolymph from each shrimp was transferred into a separatemicroncentrifuge tube held on ice. Each pool (×3) was composed ofhaemolymph from three individuals. Pooled haemolymph sampleswere filtered through an 80 µmmesh to eliminate aggregates or largepieces of debris.

2.4. Respiratory burst

To monitor the level of respiratory burst, the cell-permeant probe2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as describedby Bass et al. (1983) and Lambert et al. (2003). A volume of 2 µL DCFH-DA was added to 200 µL haemocyte samples from each of the threepools. After incubation with DCFH-DA for 30 min in the dark, themixture was diluted with 200 µL modified Alserver's solution (MAS) toobtain a final concentration of 1×106 cells/mL; it was then analyzed byusing flow cytometry. Two light-scattering parameters (forward scatterand side scatter) were used to define a gate that excluded debris andaggregates from all fluorescence analyses. For each sample, 10,000 cellswere analyzed for the two fluorescent signals (Hegaret et al., 2003). Thepercentages of cells and mean fluorescence values were calculated.

2.5. Intracellular free-Ca2+ detection by flow cytometry

Intracellular calciumconcentrationwasmonitoredusing the calciumsensitive dye fluo-3, which was dissolved in DMSO to give a stocksolution of 1mg/mLand stored at−20 °C in thedark. Fluo-3 is awidely-used, long wavelength fluorescent calcium indicator developed byProfessor Roger Tsien and colleagues (Kresge et al., 2006). The indicatorabsorbs at 526nmand canbe efficientlyexcitedbya 488 argon-ion laser.Fluo-3 is basically non-fluorescent in the absence of Ca2+, but thefluorescent signal (at 526 nm) increases by a factor of at least 40 uponCa2+ binding (Kao et al., 1989). Cell suspensions from each of the threepools were stained with fluo-3 (final concentration of 10 µM) andincubated in a 37 °C water bath for 30 min in the dark. The cells werethen centrifuged for 5 min at 700 ×g, washed and resuspended inphosphate buffer solution (PBS), filtered through a 200 µm mesh, andtransferred to flow cytometry tubes (1–2×106 cells/mL). The controlcells were incubated under identical conditions except that Fluo-3 wasabsent (Bailey and Macardle, 2006).

2.6. Single cell solution preparation

Sub-samples from each of the three pools were dilutedwith Hank'sbuffer to produce a density of 105–106 cells per mL. The hepatopan-creas from six shrimp in each group was collected after 0, 6, 12, 24 and36 h of exposure; after careful washing twice with cold physiologicalphosphate-buffered saline (pH 7.4), they were transferred to a flask.Each shrimp was tested in triplicate in the same assay. Cells from thetissues were separated by treatment with 0.1% trypsin at 37 °C for 2 h;the cells were then washed twice with PBS (pH 7.4). The final celldensity was adjusted to 2×105 cells/mL with PBS.

2.7. Single-cell gel electrophoresis—comet assay

The comet assay indicate the extent of DNA strand breaks inindividual cells bymeasuring themigration of fragmented and relaxedDNA away from the nuclei immobilized in agarose gels. The assay wascarried out according to the method described by Singh et al. (1988).An aliquot of 30 µL of the single cell suspensionwas mixed with 50 µLof 0.75% low melting point (LMP) agarose at 37 °C, and rapidly spreadonto microscope slides pre-coated with 0.5% normal melting point(NMP) agarose. Coverslips were applied and the slides were allowedto solidify at 4 °C for 10 min. The coverslips were removed gently andthe slides were then immersed in fresh cold lysing solution (2.5 M

Table 1The acute toxicity test of Cd to Litopenaeus vannamei.

[Cd2+] (µmol L−1) 8.76×10−2 0.276 0.876 2.77 8.7624 h mortality (%) 5 20 25 35 50LC50 (µmol L−1) 8.50a

a Median lethal concentrations (LC50) were calculated by probit analysis.

Fig. 2. The changes in [Ca2+]i levels in haemocytes of the pacific white shrimp exposedto different Cd2+ concentrations. Significant differences (Pb0.05) in [Ca2+]i betweenthe exposed and the control groups are indicated with asterisks. Each bar represents themean±SD (standard deviation), n=3.

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NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100, and 10%DMSO). Protected from light, the slides were maintained at 4 °C for2 h. They were then placed in electrophoresis buffer (300 mM NaOH,1 mM EDTA, pH N13) at 4 °C for 20 min before electrophoresis, toallow the DNA to unwind. After electrophoresis for 20 min at 20 V/200mA in an ice bath (4 °C), the slides were washed twicewith water,drained and stained with SYBR GOLD (1:10000) for 2 min. The cometswere analyzed at 400× magnification under an Eclipse fluorescencemicroscope (Nikon, Tokyo, Japan) attached to a digital camera (Nikon,Coolpix E995) equipped with a UV-1 filter block (an excitation filter of359 nm and a barrier filter of 461 nm). Fifty images were randomlyselected from each sample and the comet tail moment (a product ofthe fraction of DNA in the tail and the tail length) was measured usingthe PC image-analysis program CASP, as described by Konca et al.(2003). The comet tail moment is positively correlated with the levelof DNA breakage in a cell. The mean value of the tail moment in aparticular sample was used as an index of DNA damage in that sample.The length of DNA migration in the comet tail (tail length) and thepercentage of nuclear material migrating out from the comet headinto the comet tail (comet intensity) were also considered.

2.8. Statistical analyses

Quantitative data were expressed as means±SD (standarddeviation). Statistical differences were estimated by two-wayANOVA followed by Tukey's multiple range tests; the significancelevel was set at Pb0.05. All statistical analyses were performed usingSPSS 13.0 (SPSS, Chicago, IL, USA).

3. Results

The median lethal concentration (LC50) in the acute (24 h) toxicitytest was 8.50 µmol L−1 (Table 1).

Fig. 1. The changes in respiratory burst activity in haemocytes of the pacific whiteshrimp exposed to different Cd2+ concentrations. Significant differences (Pb0.05) inrespiratory burst activity between the exposed and control groups are indicated withasterisks. Each bar represents the mean±SD (standard deviation), n=3.

3.1. Respiratory burst

The changes in respiratory burst activity (determined by DCFfluorescence) in the shrimp L. vannamei haemocytes, after Cdexposure, are presented in Fig. 1. The respiratory burst activity ofshrimp haemocytes increased from 0 to 6 h regardless of the Cd2+

concentration, and then decreased to background levels after 24 h ofexposure. However, the untreated shrimp haemocytes showed nosignificant change during the treatment. The shrimps exposed to 4.25and 8.50 µmol L−1 of cadmium exhibited an increase in respiratoryburst, in comparison to the control group, after exposure for 6 h. Themean intensity of DCF fluorescence in the shrimp haemocytesexposed to 4.25 and 8.50 µmol L−1 of cadmium was 256.3±3.96and 734.38±26.20 (Pb0.05) respectively, while the value for thecontrol was 59.49±4.00 (Fig. 1).

3.2. Intracellular calcium in haemocytes

The intracellular calcium (determined by Fluo-3-AM fluorescence)in the control group remained stable throughout the 36 h exposureperiod. However, in the treatment group, the intracellular calciumlevel showed an increase after exposure for 6 h. Thereafter, asignificant decrease in calcium levels was observed for both Cdconcentrations tested. We also noticed a significant differencebetween the [Ca2+]i in shrimp haemocytes exposed to the two Cd2+

concentrations; the response was dose-dependent. The Fluo-3-AMfluorescence in the haemocytes of shrimps exposed to 4.25 and

Table 2The changes in DNA olive tail moment in the haemocytes of the shrimp L. vannameiafter exposure to Cd.

[Cd2+](µmol L−1)

The olive tail moment value in the haemocytes

0 h 6 h 12 h 24 h 36 h

0 0.36±0.23a 0.52±0.25a 0.70±0.42a 0.52±0.36a 0.22±0.10a

4.25 0.36±0.23b 9.84±3.93a⁎ 5.95±4.45a⁎ 1.22±0.49b 0.31±0.09b

8.50 0.36±0.23c 17.42±4.32a⁎ 10.92±3.11b⁎ 0.95±043c

Significant differences (Pb0.05) in the olive tail moment value (mean±SD, n=6 ) ofthe haemocytes between treatment time groups are indicated with letters (a, b, and c).Different letters indicate significant comparisons. Significant differences (Pb0.05) inthe olive tail moment of the haemocytes between the exposed and the control groupsare indicated by asterisks.

Table 3The changes in DNA olive tail moment of the hepatopancreas cells in the shrimpL. vannamei after exposure to Cd.

[Cd2+](µmol L−1)

The olive tail moment value in the hepatopancreas cells

0 h 6 h 12 h 24 h 36 h

0 0.60±0.28a 0.63±0.46a 0.84±0.41a 0.46±0.16a 0.43±0.20a

4.25 0.60±0.28c 20.21±2.66a⁎ 6.53±2.45b⁎ 1.36±0.87c 0.77±0.30c

8.50 0.60±0.28c 25.77±11.86a⁎ 8.30±3.17b⁎ 5.50±1.61c⁎

Significant differences (Pb0.05) in the olive tail moment value (mean±SD, n=6) ofthe hepatopancreas cells between treatment time groups are indicated with letters (a, b,and c). Different letters indicate significant comparisons. Significant differences(Pb0.05) in the olive tail moment of the hepatopancreas between the exposed andthe control groups are indicated by asterisks.

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8.50 µmol L−1 of cadmium was 423.62±12.93 and 712.28±8.34(Pb0.05), respectively, while the value for the control was 90.88±1.86(Fig. 2).

3.3. Comet assay

The comet parameters “Olive Tail Moment” (OTM) of the untreatedgroup remained consistent throughout the 36 h exposure period. TheDNAdamage to shrimp haemocytes and hepatopancreas cells as a resultof exposure to CdCl2 was similar. The OTM value showed an increaseafter exposure for 6 h, but a subsequent significant decrease in the OTMvalue was recorded for both concentrations tested. The shrimpsexposed to both 4.25 and 8.50 µmol L−1 of cadmium exhibited anincrease in the OTM value after exposure for 6 h compared to thecontrol group. In addition, dose-dependent DNA damage was observedin both haemocytes and the hepatopancreas after Cd treatment. Themean OTM value increased from 9.84±3.93 to 17.42±4.32 inhaemocytes and from 20.21±2.66 to 25.77±11.86 in hepatopancreascells after Cd-exposure for 6 h when the Cd2+ concentration wasincreased from 4.25 and 8.50 µmol L−1 (Tables 2 and 3; Fig. 3).

4. Discussion

Haemocytes are the cells of the open vascular system of shrimps.Since cell dissociation is not required, the degree of artificial cellulardamage from mechanical and/or proteolytic cell dissociation isminimized; in particular, low background DNA damage is expected.Moreover, haemocytes play an important physiological role inimmune defense, phagocytosis, transportation, excretion and detox-ification of xenobiotics (Siu et al., 2004). In this study, the DNAintegrity of shrimp hepatopancreas cells was assessed by the cometassay. In crustaceans, the hepatopancreas is the primary organresponsible for absorption and storage of ingested matter. Thisorgan is also involved in the accumulation and detoxification ofheavy metals (Viarengo, 1990).

Previous studies have shown that, in response to metal exposure,there is an increase in the formation of oxygen free radicals or reactiveoxygen species (ROS) in rats, rainbow trout and M. rosenbergii (Stohs

Fig. 3. The comet assay of shrimp hepatopancreas cells. The shrimp hepatopancreas cells exassay after staining with SYBR GOLD. These images of comet assay are after exposure for: 0

and Bagchi, 1995; Bopp et al., 2008; Cheng and Wang, 2001); this canresult in widespread damage to cells because of lipoperoxidation andgenotoxicity. Respiratory burst, the release of reactive oxygen species(ROS) by haemocytes, is a critical step in the innate immune responseand an important mechanism by which potential pathogens andparasites are eliminated following phagocytosis. Under normalconditions, the production and destruction of ROS is well regulated,but under environmental oxidative stress, the balance betweenprooxidative and antioxydative reactions is shifted in favor of theformer (Achard-Joris et al., 2006). Cadmium is known to displace Znand Fe ions from metalloproteins, resulting in their inactivation aswell as the release of free Fe that can then catalyze the generation ofreactive oxygen species via the Fenton reaction (Stohs and Bagchi,1995). Cd has been shown to induce ROS production in freshwatergoldfish (Carassius auratus) after exposure for 24 h (Shi et al., 2005).

Cd-promoted oxidative stress leads to DNA strand damage,especially mitochondrial DNA (Yakes and Van Houten, 1997). It isalso known that ROS can modify DNA bases and can cause strandscission by degrading the ribose ring (Yang and Gao, 2002). Althoughthere is no direct evidence yet to connect Cd-induced ROS damage toDNA strand breakage, it is plausible that mitochondrial electron flowand energy metabolismwould be disrupted during the early stages ofCd exposure, leading to mitochondrial ROS formation and oxidativedamage of mitochondrial DNA before any damage occurs in thenucleus (Watanabe et al., 2003). In this study we obtained directevidence confirming ROS generation and DNA damage after shrimpshave been exposed to Cd2+. The respiratory burst activity and DNAdamage recorded in the presence of 8.50 µmol L−1 Cd2+ was higherthan that recorded for shrimps exposed to 4.25 µmol L−1 Cd2+,suggesting that the response is dose-dependent.

Animals exposed to genotoxicants have evolved mechanisms toremove and repair DNA lesions (Hook and Lee, 2004). In this study,the intensity of respiratory burst decreased after 6 h exposure to Cd2+.Meanwhile, the degree of DNA damage also declined, suggesting thatDNA repair pathways could have been activated during this period.DNA repair pathways function to correct DNA damage that arisesspontaneously or after exposure to certain environmental agents(Hoeijmakers, 2001). The production of ROS promotes antioxidantenzyme activity, thus preventing DNA damage. Organisms havedeveloped antioxidant systems (Catalase, Glutathione peroxidase,Glutathione reductase, and Glutathione S-transferase) that provideprotection against oxidative stress damage (Downs et al., 2001).Previous studies have shown that Cd exposure induces changes inantioxidant enzyme levels and transcription (Cajaraville et al., 2003;Company et al., 2004; Lee and Shin, 2003). Gluthathione S-transferase(GST) is an important antioxidant defense enzyme that works todetoxify the products of oxidative stress. GST gene transcription andenzyme activity have been shown by other researchers (Poynton et al.,2007; Barata et al., 2005) to increase following exposure to cadmium.However, in the present study, we also observed that different fromthe respiratory activity, which decreased to basal levels after 6 h, theintracellular Ca2+ and DNA olive tail moment are still higher and

posed to 8.50 µmol L−1 were collected at different times and were subjected to cometh; 6 h; and 24 h.

585M. Chang et al. / Comparative Biochemistry and Physiology, Part C 149 (2009) 581–586

longer than the control groups. This discrepancy indicates thatprotective cellular mechanisms in response to heavy metals Cdexposure may be finite nature. The DNA damage is still present,which could be a consequence of persistent generation of DNAdamage of the failure to repair the damaged DNA. Moreover, we foundthat the degree of DNA damage in shrimps treated with 4.25 µmol L−1

Cd2+ was less than that in shrimps exposed to 8.50 µmol L−1 Cd2+.The reason might be that the shrimp L. vannamei is less affected byexposure to lower concentrations of Cd2+. It has been demonstratedthat Cd ions are able to displace zinc ions from zinc finger structures, afrequent motif in DNA repair enzymes; this would increase DNAdamage indirectly through inhibiting the repair of oxidative damagewithout any direct effect on DNA molecules (Hanas and Gunn, 1996).

It is noteworthy that DNA damage in shrimp haemocytes andhepatopancreas cells in the 1/2 24 h LC50 and 24 h LC50 (4.25 and8.50 µmol L−1, respectively) was repaired after 24 h of exposure,suggesting that Cd-induced oxidative stress may not be the mainreason for shrimp death. Cd has also been shown to induce organisminjury by inhibiting the mitochondrial electron transfer chain (Wanget al., 2004b; Achard-Joris et al., 2006).

The calcium hypothesis postulates that the basic metabolic responseto hypoxic ATP depletion is a toxic increase in free cytosolic Ca2+ in allcell types (Johnson et al., 1994). Interruption of [Ca2+]i homeostasisinduced by environmental toxicants has been reported in various celltypes. The presence of Cd could also lead to an increase in cytosolic Calevels because of its analogy with Ca (Flik et al., 1995). In addition, highconcentrations of [Ca2+]iwill strongly stimulate hydrolytic activities, forexample, endonuclease (Zirpel et al., 1998) leading to DNA fragmenta-tion and chromatin condensation. Our results show that intracellularfree calcium concentration, respiratory burst activity and DNAdamage exhibited similar patterns in response to the two testedconcentrations of cadmium, suggesting that calcium may play a majorrole during Cd-induced oxidative stress and DNA damage.

In conclusion, we have clearly demonstrated that Cd can induceoxidative stress in the shrimp L. vannamei. The results from the cometassay, respiratory burst analysis and intracellular free calciumdetermination presented here demonstrate a clear dose- and time-dependent response to Cd exposure in L. vannamei, which suggeststhat these parameters could be used as sensitive indicators (biomar-kers) for the presence of genotoxicant cadmium. We consider thisimportant evidence supporting the use of crustaceans as an indicatorof aquatic ecosystem health.

Acknowledgements

This research has been supported by the National Natural ScienceFoundation of China (30570287), and the Natural Science Foundationof Guangdong Province, PR China (06025052) (8151063101000035).We sincerely thank Prof. Chengwei Yang of South China NormalUniversity, PR China and Assistant Prof. Yanchang Wang of FloridaState University, USA for their helpful critiques of the manuscript. Thismanuscript benefited from comments provided by four anonymousreviewers.

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