research article transcriptional and biochemical effects of...

Research ArticleTranscriptional and Biochemical Effects ofCadmium and Manganese on the Defense System ofOctopus vulgaris Paralarvae

Aldo Nicosia1 Monica Salamone1 Salvatore Mazzola2 and Angela Cuttitta1

1Laboratory of Molecular Ecology and Biotechnology National Research Council Institute for Coastal Marine EnvironmentUOS Capo Granitola Torretta Granitola 91021 Trapani Italy2National Research Council Institute for Coastal Marine Environment Calata Porta di Massa 80133 Naples Italy

Correspondence should be addressed to Angela Cuttitta angelacuttittaiamccnrit

Received 2 September 2014 Revised 6 November 2014 Accepted 27 November 2014

Academic Editor Francesco Dondero

Copyright copy 2015 Aldo Nicosia et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Due to anthropogenic activities the relative concentrations of cadmium andmanganese have increased in the marine environmentCephalopods are able to accumulate such metals and as inhabitant of coastal waters Octopus vulgaris is continuously exposedto anthropogenic activities Since no study is available on the effects of heavy metals at molecular level in developing octopusesherein we exposed 1-day-old paralarvae for 24 h to 10 100 and 1000120583gL of CdCl

2orMnCl

2 Cd exerted a concentration-dependent

inhibition of survival and a reduction in growth ratewas shownwhileMn exposure did not affect the survival rate even at the highestconcentrations Gene expression profiles of hsp70 sod cat and gst genes were analyzed by quantitative real-time PCR and definedpatterns of transcription were observed Moreover posttranscriptional analyses were also performed suggesting the impairment ofmetabolic functions under strong oxidative conditions (as occurred in paralarvae exposed to Cd) or the complete detoxificationevents (as occurred in paralarvae exposed to Mn)

1 Introduction

Cadmium (Cd) and manganese (Mn) represent respectivelytypical nonessential and essential metals for the metabolismof living organisms [1]

Cd ions which enter into cells are transported to targettissues and act by a molecular and ionic mimicry mechanismsubstituting the proper ions in their metabolic sites [2] SinceCd is a permanent metal ion it is accumulated by manyorganisms which determine oxidative stress DNA damageincrease in stress proteins (hsps) and macromolecular dam-age [3 4]

Cd is also a natural constituent of ocean water withaverage levels between 5 and 20 ngL in open seas [5 6] whilehigher levels between 149 120583gL in the Galician coasts [7] and738mgL in theDardanelles Strait [8]were reported in highlypolluted coastal area

In the last century the massive production of Mn-containing compounds has attracted much interest [9] and

now Mn is considered as an emerging contaminant [10]Organisms need trace amounts of such metal [11] howeverexposure to high Mn levels is associated with genotoxic andcytotoxic effects affecting carcinogenesis and mutagenesis[12 13]

Seawater typically contains approximately 2 120583gL of Mn[14] depending on pH oxygen concentration and redoxconditions and during hypoxia it reaches concentrations upto 22mgL [15 16] The toxic effects of Cd or Mn expo-sure have been widely described in different marine organ-isms among these cephalopods are also able to accumulateessential toxic and nonessential elements [17 18] Amongcephalopods Octopus vulgaris is a cosmopolitan species thathas been recognized as an important marine resource for theworldwide commercial fisheries [19] The common octopusas inhabitant of coastal waters occupies the benthic and thepelagic zone as an adult and hatchling respectively Thus itis continuously exposed to anthropogenic activities Variousstudies reported data regarding the accumulation [20 21]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 437328 11 pageshttpdxdoiorg1011552015437328

2 BioMed Research International

and the effects of metal exposure [22 23] in tissues of adultoctopuses Although it has been shown that the developingOvulgaris eggs are able to adsorb and accumulate severalmetals[24] and in cephalopods metals affect the embryogenesis nostudy is available on the effects of heavy metals at molecularlevel in developing octopus

In a wide variety of organisms exposure to environmentalstressors such as hyperthermia hypoxia and heavy metals isassociated with the expression of heat shock proteins (HSPs)As one of the most abundant and widely investigated familiesin high eukaryotes the Hsp70 family protects the organismsfrom damage caused by an overload of unfolded proteins incells [25ndash27] Recently the role ofHSPs in the stresses defensesystem has been well documented in mollusks such asstimulated-expression of HSP70 in Mytilus galloprovincialisor Ostrea edulis by heat shock bacterial infection and metalexposures [28ndash31] Similarly catalase (CAT) superoxidedismutase (SOD) and glutathione S-transferase (GST) areknown to represent effective biomarkers of susceptibilityto environmental contaminants Their ability to counteractROS increase associated with oxidative stress is essential tocellular homeostasis [32ndash34] Moreover their transcriptionalexpression is known to be altered by metals [35ndash39]

To create new insights and to characterize the mech-anisms involved in maintaining physiological homeostasisin response to perturbation we have evaluated the effectsof metal exposure on the octopus paralarvae Herein weprovide analysis of survival rates and morphological changesin response to metals which suggest a correlation betweenmetal exposure and toxic effects Additionally a multi-genebiomarker approach was used to test the effects ofcadmium and manganese on the defense system of octopusparalarvae In this scenario the transcription of hsp70 catsod and gst was profiled in response to metal exposureMoreover the total amount ofHSP 70 protein and the activityof SOD CAT and GST were measured Both transcriptionaland biochemical studies suggest alterations of gene andprotein expressions or enzymatic activities Furthermore animpairment of the metabolic capacity in ROS removal wasalso hypothesized after exposure to cadmium excess

2 Materials and Methods

All experiments were carried out in compliance with locallaws and to date no specific permit is required for theperformed experiments Housing and husbandry of O vul-garis were carried out in accordance with the best practicesdeveloped in the cephalopods community in order to opti-mize animal health However all facilities and procedurescomplied with the Directive 201063EU

21 Animal Sampling and General Experimental Design Ovulgaris females (119899 = 4) weremanually collected in the south-west coast of Sicily over the period April-May 2012

Octopuses were acclimated at the CNR-IAMC mesoco-sms (Capo Granitola Sicily) in Millipore filtered artificial seawater (MFASW 400mMNaCl 10mMKCl 10mMCaCl

2

10mMHepes pH adjusted to 82 with NaOH) (17plusmn1∘C) andfed daily with frozen fish

Spawning occurred 30 days after capture at a temperatureof 19plusmn1∘C Eggmasseswere keptwith the female for broodingto optimise embryo survival in MFASW (constantly aeratedclosed circuit 20 plusmn 1∘C lightdark cycle 12 h12 h) until theparalarvae hatched One-day-old paralarvae were collecteddivided equally into 7 different aquaria and exposed toMnCl

2and CdCl

2in MFASW for 24 hours under gentle

rotation at 19 plusmn 1∘CCadmium and manganese solutions were prepared using

99 of chloride salts (Sigma-Aldrich)Treatments were performed using metal solutions at

nominal concentrations corresponding to 10 120583gL 100 120583gLand 1000120583gL Collected paralarvae were divided into 3parts for morphological biochemical and gene expressionanalysis Experiments were performed in triplicate

22 Description of the Morphology Morphological analysiswas carried out using an Olympus BX50 optical micro-scope Live paralarvae were sampled and anaesthetized with035mM MgCl

2 1 2 diluted with seawater A number of

50 paralarvae per treatment were scored for mortality Mea-surements of mantle (ML) and arm length (AL) were takenin both dorsal and ventral side of paralarvae Images wereanalysed using NIH-Image public domain software (version161 )

23 RNA Extraction and First-Strand cDNA Synthesis Anumber of 30 paralarvae per treatment were dissolved inTrizol reagent (Invitrogen Corporation Carlsbad USA) andfurther RNA purification steps were performed accordingto the manufacturerrsquos instructions RNA concentrations andquality were spectrophotometrically verified while RNAintegrity was checked using a 15 agarose gel The RNAwas stored at minus80∘C for future use The extracted RNA(2 120583g) was treated with RQI RNase-Free DNase (PromegaUSA) to remove any residual genomic DNA contaminationand DNase I was inactivated by adding 25mM EDTAFirst-strand cDNA was synthesised from 2120583g DNase I-treated total RNA samples using oligo(dT)

18and Superscript

III (Invitrogen Corporation Carlsbad USA) following themanufacturerrsquos instructions The cDNA mixture was storedat minus20∘C until needed

24 Gene Selection and Primer Design Genes were selectedfrom public available database at NCBIWhen not annotatedextensive BLASTP BLASTX and TBLASTN searches wereperformed to retrieve selected genes Matching sequenceswere manually checked and subjected to functional ontologyassignment based on Hidden Markov Models (HMMs)Selected full length sequences were reconfirmed by compar-ative analysis The genes specific primer sequences ampliconsizes and relative GenBank Accession Number are listed inTable 1

25 Relative Quantification Using Real-Time QuantitativePolymerase Chain Reaction (RT-qPCR) RT-qPCR was per-formed using the ABIPRISM 7500 System (Applied Biosys-tems Forster City USA) with Power Sybr Green as detectionchemistry (Applied Biosystems Forster City USA) and every

BioMed Research International 3

Table 1 Oligonucleotide primers used in this study

Gene Primers Sequences (51015840-31015840) Size (bp) GenBank

hsp70 hsp70-F CCCATAGTTAAGGGGTTGACATC 197 JR4388441hsp70-R GTGCTTGTTGGTGGCTCTACTAG

sod sod-F AGCACTCACGCATCCATTAG 156 JR4378531sod-R GGGTCTTCCGAATCTGTTTC

cat cat-F CCGTCCCTTTGATAGTTGG 114 JR4486151cat-R GGGTCGCCTGTATTCCTAC

gst gst-F AACCCAAATTCCCCAGAGTAT 129 X655431gst-R GTCGTCCAAGATGTCATAGAAGC

tubA tuba-F ACTGGTGTCCAACTGGCTTC 105 X15845tuba-R TGCTTAACATGCACACAGCA

actB act-F TGATGGCCAAGTTATCACCA 103 AB053937act-R TGGTCTCATGGATACCAGCA

18S 18S-F AGTTCCGACCGTAAACGATG 142 FJ61743918S-R CCCTTCCGTCAATTCCTTTA

ubi ubi-F TCAAAACCGCCAACTTAACC 113 FJ617440ubi-R CCTTCATTTGGTCCTTCGTC

reaction was repeated in triplicate The amplification condi-tions were initial denaturation at 95∘C for 10min 40 cyclesof 95∘C for 30 s and 60∘C for 50 s followed by a meltingcurve from 60 to 95∘C Their expression stability amongthe different conditions was evaluated using the GeNormsoftware [40] and the 18S rRNA ubi and tuba were chosenas reference genes Serial dilutions of pooled cDNAs fromboth control and treated samples were prepared to determinethe PCR efficiency of the target and reference genes (data notshown) and amplification efficiency ranged from 18 to 21A GeNorm normalization factor was calculated taking intoaccount the geometric mean of the 3 selected reference genesand used to quantify the expression levels of the target genesRelativemRNAabundances of different geneswere calculatedfrom the second derivative maximum of their respectiveamplification curves (Cp calculated by triplicates) Cp valuesfor stress-related target genes (TG) were compared to thecorresponding values for a reference gene (ref) to obtainΔCpvalues (ΔCp 14 Cpref-CpTG)

26 Protein Extraction for HSP70 Quantification Paralarvae(119899 = 30) homogenized in Lysis Buffer (05 sodium deoxy-cholate 1 NP40 01 SDS with PBS-T (phosphate bufferedsaline and 01 Tween-20 pH 75) with 2 120583g120583L antipapainleupeptin and bestatin 1 120583g120583L aprotinin and pepstatin1mM benzamidine and 01mM AEBSF) The homogenateswere centrifuged to remove any insoluble debris The super-natants were collected and dialysed against 50mM Tris-HCl(pH 75) Protein concentration was determined using theBradford assay

27 SDS-PAGE andWestern Blot Proteins (40 120583g) were sepa-rated on 8 SDS-PAGE under reducing conditions Proteinswere transferred to PVDF membrane The membrane wasthen blocked with 5 (wv) nonfat milk in TBST (50mM

Tris-HCl pH 75 150mM NaCl and 01 (vv) Tween-20 with 002 sodium azide) for 1 h at room temperatureMembranes were incubated over night at 4∘C with theprimary mouse monoclonal antibody anti-HSP70 (SigmaH5147 1 500 dilution) and with the monoclonal mouseanti-actin (Sigma A2228) The membrane was washed threetimes with TBST and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (1 8000 for 1 h at rt) Thesignals from each protein band were normalized againstthe 120573-actin content The Quantity One software (Bio-Radlaboratories) was used for densitometry analysis of theimmunoblotted bands

28 Protein Extraction for Oxidative Stress Evaluation Par-alarvae (119899 = 30 per treatment) were ground into fineparticulate using liquid nitrogen mortar and pestle Theparticulate from each sample was transferred to centrifugetube filled with 5mL of 100mM potassium phosphate bufferpH 74 150mMKCl and 01mM PMSF and centrifuged at10000timesg for 10min at 4∘C The supernatants were collectedand protein concentration was determined using the Brad-ford assay

29 Determination of Antioxidant Enzyme Activities SODactivity was measured according to McCord and Fridovich[41] and the result was expressed as unitsmg protein

CAT activity was determined according to Greenwald[42] The results were expressed as 120583mol minminus1mg protminus1

GST activity was evaluated using 1-chloro-24-dini-trobenzene (CDNB) as a substrate according to Habig etal [43] The GST activity is expressed as the 120583mol CDNBconjugate formed mgminus1 of total protein minminus1

210 Statistical Analysis Analysis was performed in tripli-cate The results were expressed as a mean value plusmn SD Data


Table 2 Survival (S) expressed as percentage of alive paralarvaeand morphological alteration (ML) expressed as mantle length ofexposed paralarvae

Level (120583gL) CdCl2 MnCl2S () ML (mm) S () ML (mm)

0 99 plusmn 2 155 plusmn 003 98 plusmn 1 155 plusmn 00310 84 plusmn 7lowast 157 plusmn 002 99 plusmn 1 155 plusmn 005100 75 plusmn 11lowast 151 plusmn 002 94 plusmn 5 157 plusmn 0021000 53 plusmn 8lowast 140 plusmn 003lowast 97 plusmn 2 165 plusmn 003lowast

Data are expressed as mean values plusmn standard error (119899 = 3) Asterisksindicate significant differences with respect to control values at 119875 lt 005

were statistically analyzed by t-test or one-way analysis ofvariance (one-way ANOVA) using Statistica 60 (StatSoftTulsa OK USA) P values less than 005 were consideredstatistically significant and were indicated by lowast

3 Results and Discussions

31 Survival Rate It has been reported that in cephalopodsmetals are transferred from the mother to the embryoshowever cadmium and manganese seemed to be retainedin the adult tissues and not transferred to the eggs [44]Thus to avoid any remaining contamination from collectedsea water paralarvae were exposed to metals in artificialsea water (MFSAW) for 24 h and nominal concentrationscorresponding to 10 120583gL 100 120583gL and 1000 120583gL of CdCl

2

or MnCl2salts were used No lethal effect was recorded

in the control group while increased CdCl2concentrations

significantly affect paralarvae lethality as they exerted toxicand concentration-dependent inhibition of the survival rate(Table 2) (119875 lt 005)

Similar results were also obtained in early life stages ofdifferent marine invertebrates including sponge polychaeteand molluscs [45ndash48]

Conversely manganese exposure did not affect the sur-vival rate and no significant mortality was observed even atthe highest concentrations (Table 2) thus paralarvae weremore sensitive to cadmium than manganese Even if limiteddata on manganese cytotoxicity in marine invertebrate sys-tems are available our results were consistent with those insea urchin embryos exposed to manganese [49 50]

32 Morphological Changes The mantle (ML) and armlength (AL) were used as indicators of growth rate (Table 2)in order to detect the presence of morphological alterationscaused by metals exposure and no significant variation in ALwas recorded (data not shown)

A negative effect in growth rate was evident in paralarvaefollowing exposure to cadmium and reduction up to 150120583min ML was found at 1000 120583gL CdCl

2(Figure 1) These fea-

tures reflect the concentration-dependent growth inhibitiondescribed previously in insect larvae of Lymantria dispar [51]and for some larvae of clam [48] and abalone [52] moreoversuch abnormalities became noticeable at 100 120583gL CdCl

2

Hence this concentration would be detrimental to octopuslarvae if such exposure was to occur

Conversely an increase in growth rate appeared followingexposure to 1000120583gL MnCl

2and paralarvae grew about

100 120583m in ML There are no studies reporting the effects onsize growth in molluscs after manganese exposure howeverthis result is anomalous if compared with the inhibitoryeffects of excess manganese described in juvenile prawnand mulloway [53 54] It is noteworthy that such reportsare confined to researches under feeding regimes Thus theeffective growth promotion exerted bymanganese in octopusparalarvae needs to be further investigated

33 Transcriptional and Biochemical Effects of Metal Expo-sure on hsp70 System The heat shock proteins have beenextensively used as stress-response marker [55 56] and theirexpression is known to be altered by metals [57 58] Thehigh mortality rate (approximately 50) occurred amongparalarvae exposed to the highest Cd exposure (1000120583gL)and the related environmental significance prompts us toanalyze transcriptional and biochemical effects exerted by Cdexclusively at 10 and 100 120583gL CdCl

2

As hypothesised a growth of hsp70 mRNA expressionwas observed following the exposure to an increasing con-centration of metals (Figure 2) and the transcript was 35-fold higher than that of the control group after exposure toCdCl2at 10 120583gL and 55-fold higher than that of the control

group after exposure to 100 120583gL Hence in the paralarvae ofO vulgaris Cd exerted concentration-dependent effects onhsp70 gene expression

Analogous results were obtained in larvae of the freshwa-ter zebra mussel Dreissena polymorpha exposed to 1 120583gL Cdfor 24 h [59] Furthermore hspA12a a member ofMytilus gal-loprovincialisHSP70 family was upregulated after exposure toCdCl25 and 50120583gL [28]

Few studies have reported the effects of manganeseexposure on the expression of hsp70 inintertidal copepodTigriopus japonicus [38] while its effects on hsp70 geneexpression have never been analysed in cephalopods Hereinwe show that hsp70 overexpression (5-fold higher than thatof the control group) was measured exclusively in responseto massive manganese presence (Figure 2) Upregulation ofhsp70 can be likely explained by the activation of HSF-HSPresponse pathway [60] Indeed it is well established andoxidative stress caused by pollutants can activate HSF toincreaseHsp70 gene expression [61]Thus in accordancewiththe absence of significant mortality during the treatmentswith Mn it could be hypothesized that a higher thresholdlevel of Mn is required to upregulate the hsp70 gene expres-sion

Genome and transcriptome sequencing efforts revealedthatmost organisms includingmollusks [62] possessmultiplemembers of the Hsp70 family some of which coexist inthe same cellular compartment Hence the expression andthe overall cellular content in HSP70 are contributed bymultiple isoforms codified by different genes existing in theparalarvaeTherefore to evaluate the effects ofmetal exposureat protein scale we analysed the total expression of HSP70 byimmunoblotting

Densitometry analysis revealed a constitutive synthesis ofthe HSP70 protein in paralarvae from the control group


Mn exposedparalarvae

Cd exposedparalarvae

Control

Figure 1 Morphological changes in response to manganese and cadmium treatments in the O vulgaris paralarvae Representative imageshowing morphological alterations (increase or reduction) in measurements of ML in paralarvae exposed to 1000120583gL CdCl

2or MnCl

2for

24 h

1

2

3

4

5

6

7

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(a)

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lowast

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expr

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MnCl2 (120583gL)

(b)

Figure 2 Expression of hsp70mRNA in response to cadmium (a) andmanganese (b) treatments in theO vulgaris paralarvaeThe expressionlevels of validated internal control genes (18S rRNA ubi and tuba) were used to calculate the normalization factor in real-time PCRexperiments Values indicate the mean plusmn SD Asterisks indicate statistically significant differences versus nonexposed controls at 119875 lt 005

Moreover indications for a concentration-dependentsynthesis of HSP70 protein was found in paralarvae exposedto Cd and Mn Data for the cadmium-induced HSP70response in aquatic molluscs are limited however in isolatedgill and hepatopancreas cells of the eastern oysterCrassostreavirginica exposure to cadmium resulted in a dose-dependentincrease of HSP70 proteins and in blue musselMytilus edulisHSP70 represents indicator of heavy metals accumulation[58 63]

Similarly herein we show that octopus paralarvae syn-thesize HSP70 protein as a general protective factor even inresponse to the lowest cadmium exposure

Analysis of HSP70 protein expression was also per-formed in manganese exposed paralarvae and a differentprofile was revealed A mild increase in the amount of totalHSP70 proteins was exclusively reported when paralarvaewere subjected to 1000120583gL MnCl

2(Figure 3) Therefore

in octopuses HSP70s would not respond against relativelow concentrations of manganese while it was moderatelyinduced when the highest concentration was achieved

In this context the expression profile of HSP70 appearsto resemble its homologue in other species including Para-centrotus lividus embryos [50] Considering these results theubiquitous nature of HSP70 family and activities it appearsthat expression of HSP70s represents a general feature of thecellular response tometals across the different taxa includingearly life stages

34 Transcriptional and Biochemical Effects of Metal Expo-sure on Antioxidant System The mRNA levels of the threeselected genes (sod cat and gst) potentially involved in cellstress defense and antioxidant mechanisms are shown inFigure 4

The relative expression of sod cat and gst increasedin paralarvae exposed to Cd reaching its peak at 100 120583gLCdCl2 In particular sod expression was elevated to approx-

imately 5-fold greater than the control cat mRNA accumu-lated to 35-fold greater than the control and gst transcriptwas overexpressed approximately 6-fold greater than the con-trol group


0

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lowast

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0 1000100100 10010

HSP70

HSP70 protein expressionHSP70 protein expressionFo

ld in

crea

se

Fold

incr

ease

120573-Actin

CdCl2 (120583gL)

CdCl2 (120583gL)

MnCl2 (120583gL)

MnCl2 (120583gL)

(a) (b)

Figure 3 Effects of cadmium (a) and manganese (b) exposure on expression of HSP70 heat shock protein in O vulgaris paralarvaeRepresentative immunoblot of HSP70 in paralarvae collected 24 h after exposure to CdCl

2and MnCl

2at the indicated concentrations The

histograms represent the average fold increase values plusmn SD of HSP70 calculated after normalization with actin levels Asterisks indicatestatistically significant differences versus nonexposed controls at 119875 lt 005

Our data correspond to other works showing the induc-tion of antioxidant genes in fish [64 65] exposed to Cd

Additionally similar results were obtained in Crassostreagigas [36]Therefore the expression of the mRNA for antiox-idant genes suggests that ROS were induced by Cd in par-alarvae and that the antioxidant system was transcriptionallyenhanced to remove the ROS

The transcriptional activity of these genes in response toMn did not show such a huge variation Indeed the relativeexpression of sod cat and gst peaked exclusively in paralarvaeexposed to 1000 120583gL MnCl

2

Even though there are limited data available on tran-scriptional effects exerted by Mn exposure our findings arein accordance with the results from the copepods Tigriopusjaponicus exposed to Mn [38 66]

The analysis of mRNA levels allowed us to hypothesizealso a coordinate expression of stressoxidative metabolism-related genes Indeed the sod cat and gst mRNA levelsappeared mutually correlated in paralarvae exposed to bothmetals a finding which is consistent with their relativelysimilar response to metal treatments

These genes are under the control of antioxidant responseelements (AREs) in many organisms [67] and their coordi-nate expression may be associated with detoxification events

in response to variations of the redox statusThus it could behypothesized that putative transcription factors coregulatingthese genes (likely through ARE or similar controlling ele-ments in their promoter sequences) are already present at thelarval stage

The activity of SOD CAT and GST enzymes was alsoprofiled in order to evaluate the general status of antiox-idant system and to test the presumptive impairment ofthe metabolic capacity The SOD activity increased in par-alarvae collected after 10 120583gL and 100 120583gL CdCl

2exposure

(Figure 5(a)) Conversely increasing in SOD activity wasfound in paralarvae after exposure to 100 and 1000120583gLMnCl

2(Figure 5(b))

Concerning the CAT activity a dose-dependent increasewas found in paralarvae collected after 10 and 100 120583gLCdCl2treatments (Figure 6(a)) MnCl

2exposure differently

affected the CAT activity indeed it was considerably highercompared to controlsin response to 10120583gL and 100 120583gLMnCl

2exposure while it dropped markedly in paralarvae

exposed to 1000 120583gL MnCl2(Figure 6(b))

Lastly GST activity was similarly affected by CdCl2and

MnCl2since it increased in response to 10 120583gL and 100 120583gL

while a decrease was measured in paralarvae exposed to1000 120583gL MnCl

2(Figure 7)


0 10010 0 10010 0 10010

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(a)

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lowast

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(b)

Figure 4 Expression of antioxidant enzymes mRNA in response to cadmium (a) and manganese (b) treatment in the O vulgaris paralarvaeThe expression levels of 3 validated internal control genes (18S rRNA ubi and tuba) were used to calculate the normalization factor in real-time PCR experiments Values indicate the mean plusmn SD Asterisks indicate statistically significant differences versus nonexposed controls at119875 lt 005

0

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activ

ity lowast

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(a)

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activ

ity

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(b)

Figure 5 Superoxide dismutase activity in O vulgaris paralarvae exposed to cadmium (a) and manganese (b) Paralarvae were treated with0 (control) 10 and 100 120583gL CdCl

2or 10 100 and 1000 120583gL MnCl

2for 24 hours All data are represented as mean plusmn SD Asterisks indicate

statistically significant differences versus nonexposed controls at 119875 lt 005


0

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tivity

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(a)

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activ

ity

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lowast

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(b)

Figure 6 Catalase activity in O vulgaris paralarvae exposed to cadmium (a) and manganese (b) Paralarvae were treated with 0 (control)10 and 100 120583gL CdCl

2or 10 100 and 1000 120583gL MnCl

2for 24 hours All data are represented as mean plusmn SD (30 paralarvae per treatment)

Asterisks indicate statistically significant differences versus nonexposed controls at 119875 lt 005

0

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activ

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(a)

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activ

ity

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(b)

Figure 7 Glutathione S-transferases activity in O vulgaris paralarvae exposed to cadmium (a) and manganese (b) Paralarvae were treatedwith 0 (control) 10 and 100120583gL CdCl

2or 10 100 and 1000 120583gL MnCl

2for 24 hours All data are represented as mean plusmn SD Asterisks

indicate statistically significant differences versus nonexposed controls at 119875 lt 005

Considering the production of ROS caused by metalexposure the activity of antioxidant enzymes is usuallyenhanced [32 68 69] Thus it is reasonable to hypothe-size that in octopus paralarvae the activity of antioxidantenzymes was enhanced to restore physiological homeosta-sis At relatively low or medium metal concentrations theROS production in paralarvae may be nullified by theincreased SOD CAT and GST activities and it may be

considered as a general protective strategy to avoid toxic-ity

Conversely metal excess resulted in an antioxidant activ-ities reduction or inhibition A reduction in antioxidantactivities was also reported in early life stages of severalorganisms [70 71] after exposure to metal stress

Thus even if the defense mechanisms are activated thehuge toxicity exerted by cadmium beyond a certain level


(1000 120583gL) results in a decrease of antioxidant function (datanot shown) that is harmful to the survival of paralarvae andmay be associated with an impairment of metabolic capacity

Additionally low levels of antioxidant enzymes inmusselshave been interpreted as a sign of susceptibility to oxidativestress [72 73]

Therefore we deduced that the antioxidant dropping offmay be associated with complete detoxification events (aslikely occurred in paralarvae exposed to Mn) or with theimpairment of metabolic functions under strong oxidativestress which in turn resulted in the inability to restore thephysiological homeostasis (as occurred in paralarvae exposedto Cd)

4 Conclusions

O vulgaris is one of the most economically valuable speciesamong cephalopods and the intake of essential and nonessen-tial elements from the increasing consumption of octopusflesh may represent a potential risk of human exposure

In the present study concentrations from 10 to 1000120583gLof both metals were used they were selected and used asthey likely represent environmentally relevant albeit ele-vated exposureMoreover the concentrations herein used aresimilar to those tested in other studies [37 38 74 75]

Data herein presented strongly suggest a different tol-erance of the paralarvae towards cadmium and manganeseCadmium has an oxidative stress potential greater thanmanganese which resulted in hazardous effect and mortalityalso at relatively low concentrations Additionally to thebest of our knowledge herein we provide the first efforttowards the depicting and understanding of the transcrip-tional and biochemical mechanisms of the response againstmetal exposures in the juvenile octopuses Moreover thiswork represents the first report on the effects of Mn inmolluscs Our results indicated that Cd and Mn exposurecould affect stress and detoxification related pathways at geneexpression protein and enzymatic level in O vulgaris

However the expression profile analysis of antioxidativestress genes in paralarvae needs to be further investigatedThus a comprehensive mining and profiling of the hsp70 sodcat and gst gene families could provide a better understand-ing of the molecular mechanisms of metal-induced cellulardamage in juvenile octopus

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank John Cicchirillo for the experience in thebreeding and domestication ofOctopus vulgaris and CarmeloBennici and Tiziana Masullo for the experimental supportFunds were provided by Ritmare Flagsheet Project to AngelaCuttitta

References

[1] E C Foulkes ldquoTransport of toxic heavymetals across cell mem-branes A reviewrdquo Proceedings of the Society for ExperimentalBiology and Medicine vol 223 pp 234ndash240 2000

[2] C C Bridges and R K Zalups ldquoMolecular and ionic mimicryand the transport of toxic metalsrdquo Toxicology and AppliedPharmacology vol 204 no 3 pp 274ndash308 2005

[3] M Shimizu J F Hochadel and M P Waalkes ldquoEffects ofglutathione depletion on cadmium-induced metallothioneinsynthesis cytotoxicity and proto-oncogene expression in cul-tured rat myoblastsrdquo Journal of Toxicology and EnvironmentalHealth vol 51 no 6 pp 609ndash621 1997

[4] N Ercal H Gurer-Orhan and N Aykin-Burns ldquoToxic metalsand oxidative stress part I mechanisms involved in metal-induced oxidative damagerdquo Current Topics in Medicinal Chem-istry vol 1 no 6 pp 529ndash539 2001

[5] OSPARCadmium Hazardous Substances Series OSPARCom-mission 2002

[6] K Kremling and P Streu ldquoBehaviour of dissolved Cd Co Znand Pb in North Atlantic near-surface waters (30∘ N60∘ W to60∘N2∘W)rdquoDeep SeaResearch Part I vol 4812 pp 2541ndash25672001

[7] R Beiras J Bellas N Fernandez J I Lorenzo and A Cobelo-Garcıa ldquoAssessment of coastal marine pollution in Galicia (NWIberian Peninsula)metal concentrations in seawater sedimentsand mussels (Mytilus galloprovincialis) versus embryo-larvalbioassays using Paracentrotus lividus and Ciona intestinalisrdquoMarine Environmental Research vol 56 no 4 pp 531ndash553 2003

[8] S Yılmaz and M Sadikoglu ldquoStudy of heavy metal pollution inseawater of Kepez harbor of Canakkale (Turkey)rdquo Environmen-tal Monitoring and Assessment vol 173 no 1ndash4 pp 899ndash9042011

[9] Y V V Satyanarayana and R Saraf ldquoIron and manganesecontamination sources adverse effects and control methodsrdquoJournal of Environmental Science and Engineering vol 49 no 4pp 333ndash336 2007

[10] CICAD ldquoManganese and its compounds environmental as-pectsrdquo Concise International Chemical Assessment Document63 World Health Organization Geneva Switzerland 2004

[11] A B Santamaria ldquoManganese exposure essentiality and toxic-ityrdquo Indian Journal of Medical Research vol 128 no 4 pp 484ndash500 2008

[12] G B Gerber A Leonard and P Hantson ldquoCarcinogenicitymutagenicity and teratogenicity of manganese compoundsrdquoCritical Reviews in OncologyHematology vol 42 no 1 pp 25ndash34 2002

[13] P D L Lima M C Vasconcellos M O Bahia et al ldquoGenotoxicand cytotoxic effects of manganese chloride in cultured humanlymphocytes treated in different phases of cell cyclerdquo Toxicologyin Vitro vol 22 no 4 pp 1032ndash1037 2008

[14] R N McNeely V P Neimanis and L Dwyer Water QualitySourcebook A Guide to Water Quality Parameters InlandWaters Directorate Environment Canada 1979

[15] J H Trefry B J Presley W L Keeney-Kennicutt and R PTrocine ldquoDistribution and chemistry of manganese iron andsuspended particulates in orca basinrdquo Geo-Marine Letters vol4 no 2 pp 125ndash130 1984

[16] R C Aller ldquoThe sedimentary Mn cycle in long island sound itsrole as intermediate oxidant and the influence of bioturbationO2 and Corg flux on diagenetic reaction balancesrdquo Journal of

Marine Research vol 52 no 2 pp 259ndash295 1994


[17] P Miramand and J C Guary ldquoHigh concentrations of someheavy metals in tissues of the mediterranean octopusrdquo Bulletinof Environmental Contamination and Toxicology vol 24 no 1pp 783ndash788 1980

[18] R Villanueva and P Bustamante ldquoComposition in essentialand non-essential elements of early stages of cephalopods anddietary effects on the elemental profiles of Octopus vulgarisparalarvaerdquo Aquaculture vol 261 no 1 pp 225ndash240 2006

[19] A Guerra ldquoOctopus vulgaris a review of the world fisheryrdquo inProceedings Workshop on the Fishery and Market Potential ofOctopus in California M A Lang and F G Hochberg Eds pp91ndash98 Smithsonian Institution Washington DC USA 1997

[20] J Raimundo M Caetano and C Vale ldquoGeographical variationand partition of metals in tissues of Octopus vulgaris along thePortuguese coastrdquo Science of the Total Environment vol 325 no1ndash3 pp 71ndash81 2004

[21] S Seixas P Bustamante and G J Pierce ldquoInterannual patternsof variation in concentrations of trace elements in arms ofOctopus vulgarisrdquo Chemosphere vol 59 no 8 pp 1113ndash11242005

[22] J Raimundo C Vale R Duarte and I Moura ldquoSub-cellularpartitioning of Zn Cu Cd and Pb in the digestive gland ofnative Octopus vulgaris exposed to different metal concentra-tions (Portugal)rdquo Science of the Total Environment vol 390 no2-3 pp 410ndash416 2008

[23] M Semedo M A Reis-Henriques L Rey-Salgueiro et alldquoMetal accumulation andoxidative stress biomarkers in octopus(Octopus vulgaris) fromNorthwest Atlanticrdquo Science of the TotalEnvironment vol 433 pp 230ndash237 2012

[24] T Lacoue-Labarthe R Villanueva C Rouleau et al ldquoRadioiso-topes demonstrate the contrasting bioaccumulation capacitiesof heavy metals in embryonic stages of cephalopod speciesrdquoPLoS ONE vol 6 no 11 Article ID e27653 2011

[25] J Ellis ldquoProteins as molecular chaperonesrdquoNature vol 328 no6129 pp 378ndash379 1988

[26] M-J Gething and J Sambrook ldquoProtein folding in the cellrdquoNature vol 355 no 6355 pp 33ndash45 1992

[27] M-F Tsan and B Gao ldquoHeat shock protein and innate immu-nityrdquo Cellular amp Molecular Immunology vol 1 no 4 pp 274ndash279 2004

[28] L You X Ning F Liu J Zhao Q Wang and H WuldquoThe response profiles of HSPA12A and TCTP from Mytilusgalloprovincialis to pathogen and cadmium challengerdquo Fish andShellfish Immunology vol 35 no 2 pp 343ndash350 2013

[29] C Cellura M Toubiana N Parrinello and P Roch ldquoHSP70gene expression in Mytilus galloprovincialis hemocytes is trig-gered by moderate heat shock and Vibrio anguillarum but notby V splendidus or Micrococcus lysodeikticusrdquo Developmentaland Comparative Immunology vol 30 no 11 pp 984ndash997 2006

[30] A Piano P Valbonesi and E Fabbri ldquoExpression of cytopro-tective proteins heat shock protein 70 and metallothioneins intissues of Ostrea edulis exposed to heat and heavy metalsrdquo CellStress amp Chaperones vol 9 no 2 pp 134ndash142 2004

[31] M C Roccheri M Agnello R Bonaventura and V MatrangaldquoCadmium induces the expression of specific stress proteinsin sea urchin embryosrdquo Biochemical and Biophysical ResearchCommunications vol 321 no 1 pp 80ndash87 2004

[32] D R Livingstone ldquoContaminant-stimulated reactive oxygenspecies production and oxidative damage in aquatic organismsrdquoMarine Pollution Bulletin vol 42 no 8 pp 656ndash666 2001

[33] V Funes J Alhama J I Navas J Lopez-Barea and J PeinadoldquoEcotoxicological effects of metal pollution in two molluscspecies from the Spanish South Atlantic littoralrdquo EnvironmentalPollution vol 139 no 2 pp 214ndash223 2006

[34] A Valavanidis T VlahogianniMDassenakis andM ScoullosldquoMolecular biomarkers of oxidative stress in aquatic organismsin relation to toxic environmental pollutantsrdquoEcotoxicology andEnvironmental Safety vol 64 no 2 pp 178ndash189 2006

[35] R Chandran A A Sivakumar S Mohandass and MAruchami ldquoEffect of cadmium and zinc on antioxidant enzymeactivity in the gastropod Achatina fulicardquo Comparative Bio-chemistry and PhysiologymdashC Toxicology and Pharmacology vol140 no 3-4 pp 422ndash426 2005

[36] P G Jo Y K Choi and C Y Choi ldquoCloning and mRNAexpression of antioxidant enzymes in the Pacific oyster Cras-sostrea gigas in response to cadmium exposurerdquo ComparativeBiochemistry and Physiology C Toxicology amp Pharmacology vol147 no 4 pp 460ndash469 2008

[37] D A Taylor E L Thompson S V Nair and D A RaftosldquoDifferential effects of metal contamination on the transcriptexpression of immune- and stress-response genes in the SydneyRock oyster Saccostrea glomeratardquoEnvironmental Pollution vol178 pp 65ndash71 2013

[38] B-M Kim B-S Choi K-W Lee et al ldquoExpression profileanalysis of antioxidative stress and developmental pathwaygenes in the manganese-exposed intertidal copepod Tigriopusjaponicus with 6K oligochiprdquo Chemosphere vol 92 no 9 pp1214ndash1223 2013

[39] M Gonzalez-Rey J J Mattos C E Piazza A C D Bainy andM J Bebianno ldquoEffects of active pharmaceutical ingredientsmixtures in mussel Mytilus galloprovincialisrdquo Aquatic Toxicol-ogy vol 153 pp 12ndash26 2014

[40] J Vandesompele K De Preter F Pattyn et al ldquoAccurate nor-malization of real-time quantitative RT-PCR data by geometricaveraging of multiple internal control genesrdquo Genome Biologyvol 3 no 7 Article ID RESEARCH0034 2002

[41] J M McCord and I Fridovich ldquoSuperoxide dismutase anenzymic function for erythrocuprein (hemocuprein)rdquoThe Jour-nal of Biological Chemistry vol 244 no 22 pp 6049ndash6055 1969

[42] R A Greenwald Handbook of Methods for Oxygen RadicalResearch CRC Press Boca Raton Fla USA 1985

[43] W H Habig M J Pabst and W B Jakoby ldquoGlutathione-s-transferases the first enzymatic step in mercapturic acidformationrdquoThe Journal of Biological Chemistry vol 249 no 22pp 7130ndash7139 1974

[44] T Lacoue-Labarthe M Warnau F Oberhansli J-L TeyssieN Koueta and P Bustamante ldquoDifferential bioaccumulationbehaviour of Ag and Cd during the early development of thecuttlefish Sepia officinalisrdquoAquatic Toxicology vol 86 no 3 pp437ndash446 2008

[45] M Coeurdassier A de Vaufleury R Scheifler E Morhain andP-M Badot ldquoEffects of cadmium on the survival of three life-stages of the freshwater pulmonate Lymnaea stagnalis (Mol-lusca Gastropoda)rdquo Bulletin of Environmental Contaminationand Toxicology vol 72 no 5 pp 1083ndash1090 2004

[46] H Sen and U Sunlu ldquoEffects of Cadmium (CdCl2) on develop-

ment and hatching of eggs in European squid (Loligo vulgarisLamarck 1798) (Cephalopoda Loliginidae)rdquo EnvironmentalMonitoring and Assessment vol 133 no 1ndash3 pp 371ndash378 2007

[47] M Ansaldo D E Nahabedian C Di Fonzo and E A WiderldquoEffect of cadmium lead and arsenic on the oviposition


hatching and embryonic survival of Biomphalaria glabratardquoScience of the Total Environment vol 407 no 6 pp 1923ndash19282009

[48] QWang B Liu H Yang XWang and Z Lin ldquoToxicity of leadcadmium andmercury on embryogenesis survival growth andmetamorphosis ofMeretrix meretrix larvaerdquo Ecotoxicology vol18 no 7 pp 829ndash837 2009

[49] N Kobayashi and H Okamura ldquoEffects of heavy metals on seaurchin embryo development Part 2 Interactive toxic effects ofheavy metals in synthetic mine effluentsrdquo Chemosphere vol 61no 8 pp 1198ndash1203 2005

[50] A Pinsino V Matranga F Trinchella and M C RoccherildquoSea urchin embryos as an in vivo model for the assessment ofmanganese toxicity developmental and stress response effectsrdquoEcotoxicology vol 19 no 3 pp 555ndash562 2010

[51] D Mircic D Blagojevic V Peric-Mataruga et al ldquoCadmiumeffects on the fitness-related traits and antioxidative defense ofLymantria dispar L larvaerdquoEnvironmental Science andPollutionResearch vol 20 no 1 pp 209ndash218 2013

[52] J Gorski and D Nugegoda ldquoSublethal toxicity of trace metalsto larvae of the blacklip abaloneHaliotis rubrardquo EnvironmentalToxicology and Chemistry vol 25 no 5 pp 1360ndash1367 2006

[53] S Adhikari A A Naqvi C Pani B R Pillai J K Jena and NSarangi ldquoEffect of manganese and iron on growth and feedingof Juvenile Giant River PrawnMacrobrachium rosenbergii (De-Man)rdquo Journal of the World Aquaculture Society vol 38 no 1pp 161ndash168 2007

[54] G J Partridge and A J Lymbery ldquoEffects of manganese onjuvenile mulloway (Argyrosomus japonicus) cultured in waterwith varying salinity-implications for inland mariculturerdquoAquaculture vol 290 no 3-4 pp 311ndash316 2009

[55] M E Clayton R Steinmann and K Fent ldquoDifferent expressionpatterns of heat shock proteins hsp 60 and hsp 70 in zebra mus-sels (Dreissena polymorpha) exposed to copper and tributyltinrdquoAquatic Toxicology vol 47 no 3-4 pp 213ndash226 2000

[56] Q Gao L Song D Ni L Wu H Zhang and Y ChangldquocDNA cloning and mRNA expression of heat shock protein90 gene in the haemocytes of Zhikong scallop Chlamys farrerirdquoComparative Biochemistry and Physiology Part B Biochemistryand Molecular Biology vol 147 no 4 pp 704ndash715 2007

[57] Y K Choi P G Jo and C Y Choi ldquoCadmium affectsthe expression of heat shock protein 90 and metallothioneinmRNA in the Pacific oyster Crassostrea gigasrdquo ComparativeBiochemistry and PhysiologymdashC Toxicology and Pharmacologyvol 147 no 3 pp 286ndash292 2008

[58] A V Ivanina A S Cherkasov and I M Sokolova ldquoEffectsof cadmium on cellular protein and glutathione synthesis andexpression of stress proteins in eastern oysters Crassostreavirginica GmelinrdquoThe Journal of Experimental Biology vol 211no 4 pp 577ndash586 2008

[59] A Navarro M Faria C Barata and B Pina ldquoTranscriptionalresponse of stress genes to metal exposure in zebra mussellarvae and adultsrdquo Environmental Pollution vol 159 no 1 pp100ndash107 2011

[60] R I Morimoto ldquoCells in stress transcriptional activation ofheat shock genesrdquo Science vol 259 no 5100 pp 1409ndash14101993

[61] Y J Lee and P M Corry ldquoMetabolic oxidative stress-inducedHSP70 gene expression is mediated through SAPK pathwayrole of Bcl-2 and c-Jun NH

2-terminal kinaserdquo The Journal of

Biological Chemistry vol 273 no 45 pp 29857ndash29863 1998

[62] E Fabbri P Valbonesi and S Franzellitti ldquoHSP expression inbivalvesrdquo Invertebrate Survival Journal vol 5 pp 135ndash161 2008

[63] M Radlowska and J Pempkowiak ldquoStress-70 as indicator ofheavy metals accumulation in blue mussel Mytilus edulisrdquoEnvironment International vol 27 no 8 pp 605ndash608 2002

[64] M Romeo N Bennani M Gnassia-Barelli M Lafaurie andJ P Girard ldquoCadmium and copper display different responsestowards oxidative stress in the kidney of the sea bass Dicentrar-chus labraxrdquo Aquatic Toxicology vol 48 no 2-3 pp 185ndash1942000

[65] B H Hansen S Roslashmma Oslash A Garmo P A Olsvik andR A Andersen ldquoAntioxidative stress proteins and their geneexpression in brown trout (Salmo trutta) from three rivers withdifferent heavy metal levelsrdquo Comparative Biochemistry andPhysiology Part C Toxicology and Pharmacology vol 143 no 3pp 263ndash274 2006

[66] Y-M Lee K-W Lee H Park et al ldquoSequence biochemicalcharacteristics and expression of a novel Sigma-class of glu-tathione S-transferase from the intertidal copepod Tigriopusjaponicus with a possible role in antioxidant defenserdquo Chemo-sphere vol 69 no 6 pp 893ndash902 2007

[67] C I Jones III H Zhu S F Martin Z Han Y Li and B RAlevriadou ldquoRegulation of antioxidants and phase 2 enzymesby shear-induced reactive oxygen species in endothelial cellsrdquoAnnals of Biomedical Engineering vol 35 no 5 pp 683ndash6932007

[68] H Kappus ldquoLipid peroxidation mechanisms analysis enzy-mology and biological relevancerdquo in Oxidative Stress H SiesEd pp 273ndash310 Academic Press London UK 1985

[69] GWWinston and R T Di Giulio ldquoProoxidant and antioxidantmechanisms in aquatic organismsrdquo Aquatic Toxicology vol 19no 2 pp 137ndash161 1991

[70] L D Peters and D R Livingstone ldquoAntioxidant enzymeactivities in embryologic and early larval stages of turbotrdquoJournal of Fish Biology vol 49 no 5 pp 986ndash997 1996

[71] L Cao W Huang J Liu X Yin and S Dou ldquoAccumulationand oxidative stress biomarkers in Japanese flounder larvae andjuveniles under chronic cadmium exposurerdquo Comparative Bio-chemistry and Physiology Part C Toxicology amp Pharmacologyvol 151 no 3 pp 386ndash392 2010

[72] A M Osman H van den Heuvel and P C M van NoortldquoDifferential responses of biomarkers in tissues of a freshwatermussel Dreissena polymorpha to the exposure of sedimentextracts with different levels of contaminationrdquo Journal ofApplied Toxicology vol 27 no 1 pp 51ndash59 2007

[73] B Fernandez J A Campillo C Martınez-Gomez and JBenedicto ldquoAssessment of the mechanisms of detoxification ofchemical compounds and antioxidant enzymes in the digestivegland of musselsMytilus galloprovincialis fromMediterraneancoastal sitesrdquo Chemosphere vol 87 no 11 pp 1235ndash1245 2012

[74] A V Hallare M Schirling T Luckenbach H-R Kohler andR Triebskorn ldquoCombined effects of temperature and cadmiumon developmental parameters and biomarker responses inzebrafish (Danio rerio) embryosrdquo Journal of Thermal Biologyvol 30 no 1 pp 7ndash17 2005

[75] R Chandurvelan I DMarsden S Gaw andC N Glover ldquoBio-chemical biomarker responses of green-lipped mussel Pernacanaliculus to acute and subchronic waterborne cadmiumtoxicityrdquo Aquatic Toxicology vol 140-141 pp 303ndash313 2013

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


and the effects of metal exposure [22 23] in tissues of adultoctopuses Although it has been shown that the developingOvulgaris eggs are able to adsorb and accumulate severalmetals[24] and in cephalopods metals affect the embryogenesis nostudy is available on the effects of heavy metals at molecularlevel in developing octopus

In a wide variety of organisms exposure to environmentalstressors such as hyperthermia hypoxia and heavy metals isassociated with the expression of heat shock proteins (HSPs)As one of the most abundant and widely investigated familiesin high eukaryotes the Hsp70 family protects the organismsfrom damage caused by an overload of unfolded proteins incells [25ndash27] Recently the role ofHSPs in the stresses defensesystem has been well documented in mollusks such asstimulated-expression of HSP70 in Mytilus galloprovincialisor Ostrea edulis by heat shock bacterial infection and metalexposures [28ndash31] Similarly catalase (CAT) superoxidedismutase (SOD) and glutathione S-transferase (GST) areknown to represent effective biomarkers of susceptibilityto environmental contaminants Their ability to counteractROS increase associated with oxidative stress is essential tocellular homeostasis [32ndash34] Moreover their transcriptionalexpression is known to be altered by metals [35ndash39]

To create new insights and to characterize the mech-anisms involved in maintaining physiological homeostasisin response to perturbation we have evaluated the effectsof metal exposure on the octopus paralarvae Herein weprovide analysis of survival rates and morphological changesin response to metals which suggest a correlation betweenmetal exposure and toxic effects Additionally a multi-genebiomarker approach was used to test the effects ofcadmium and manganese on the defense system of octopusparalarvae In this scenario the transcription of hsp70 catsod and gst was profiled in response to metal exposureMoreover the total amount ofHSP 70 protein and the activityof SOD CAT and GST were measured Both transcriptionaland biochemical studies suggest alterations of gene andprotein expressions or enzymatic activities Furthermore animpairment of the metabolic capacity in ROS removal wasalso hypothesized after exposure to cadmium excess

2 Materials and Methods

All experiments were carried out in compliance with locallaws and to date no specific permit is required for theperformed experiments Housing and husbandry of O vul-garis were carried out in accordance with the best practicesdeveloped in the cephalopods community in order to opti-mize animal health However all facilities and procedurescomplied with the Directive 201063EU

21 Animal Sampling and General Experimental Design Ovulgaris females (119899 = 4) weremanually collected in the south-west coast of Sicily over the period April-May 2012

Octopuses were acclimated at the CNR-IAMC mesoco-sms (Capo Granitola Sicily) in Millipore filtered artificial seawater (MFASW 400mMNaCl 10mMKCl 10mMCaCl

2

10mMHepes pH adjusted to 82 with NaOH) (17plusmn1∘C) andfed daily with frozen fish

Spawning occurred 30 days after capture at a temperatureof 19plusmn1∘C Eggmasseswere keptwith the female for broodingto optimise embryo survival in MFASW (constantly aeratedclosed circuit 20 plusmn 1∘C lightdark cycle 12 h12 h) until theparalarvae hatched One-day-old paralarvae were collecteddivided equally into 7 different aquaria and exposed toMnCl

2and CdCl

2in MFASW for 24 hours under gentle

rotation at 19 plusmn 1∘CCadmium and manganese solutions were prepared using

99 of chloride salts (Sigma-Aldrich)Treatments were performed using metal solutions at

nominal concentrations corresponding to 10 120583gL 100 120583gLand 1000120583gL Collected paralarvae were divided into 3parts for morphological biochemical and gene expressionanalysis Experiments were performed in triplicate

22 Description of the Morphology Morphological analysiswas carried out using an Olympus BX50 optical micro-scope Live paralarvae were sampled and anaesthetized with035mM MgCl

2 1 2 diluted with seawater A number of

50 paralarvae per treatment were scored for mortality Mea-surements of mantle (ML) and arm length (AL) were takenin both dorsal and ventral side of paralarvae Images wereanalysed using NIH-Image public domain software (version161 )

23 RNA Extraction and First-Strand cDNA Synthesis Anumber of 30 paralarvae per treatment were dissolved inTrizol reagent (Invitrogen Corporation Carlsbad USA) andfurther RNA purification steps were performed accordingto the manufacturerrsquos instructions RNA concentrations andquality were spectrophotometrically verified while RNAintegrity was checked using a 15 agarose gel The RNAwas stored at minus80∘C for future use The extracted RNA(2 120583g) was treated with RQI RNase-Free DNase (PromegaUSA) to remove any residual genomic DNA contaminationand DNase I was inactivated by adding 25mM EDTAFirst-strand cDNA was synthesised from 2120583g DNase I-treated total RNA samples using oligo(dT)

18and Superscript

III (Invitrogen Corporation Carlsbad USA) following themanufacturerrsquos instructions The cDNA mixture was storedat minus20∘C until needed

24 Gene Selection and Primer Design Genes were selectedfrom public available database at NCBIWhen not annotatedextensive BLASTP BLASTX and TBLASTN searches wereperformed to retrieve selected genes Matching sequenceswere manually checked and subjected to functional ontologyassignment based on Hidden Markov Models (HMMs)Selected full length sequences were reconfirmed by compar-ative analysis The genes specific primer sequences ampliconsizes and relative GenBank Accession Number are listed inTable 1

25 Relative Quantification Using Real-Time QuantitativePolymerase Chain Reaction (RT-qPCR) RT-qPCR was per-formed using the ABIPRISM 7500 System (Applied Biosys-tems Forster City USA) with Power Sybr Green as detectionchemistry (Applied Biosystems Forster City USA) and every





























2










2






2










Control


2or MnCl

2for

24 h

1

2

3

4

5

6

7

00 10010


lowast

lowast

Relat

ive m

RNA

expr

essio

n

CdCl2 (120583gL)

(a)

1

2

3

4

5

6

7

00 10 100 1000


lowast

Relat

ive m

RNA

expr

essio

n

MnCl2 (120583gL)

(b)












0

1

2

3

4

0

1

2

3

4

lowast

lowast

0 10010 0 100010010

0 1000100100 10010

HSP70


ld in

crea

se

Fold

incr

ease

120573-Actin

CdCl2 (120583gL)

CdCl2 (120583gL)

MnCl2 (120583gL)

MnCl2 (120583gL)

(a) (b)


2and MnCl






2






2exposure


2(Figure 5(b))









2(Figure 7)


0 10010 0 10010 0 10010

lowast

lowast

1

2

3

4

5

6

7

lowastlowast

lowast

1

2

3

4

5

6

7

1

2

3

4

5

6

7


0 0 0

sod cat gst

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(a)

0 100010010 0 100010010 0 100010010

lowast

0

lowast

lowast

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(b)


0

3

6

9

12

15

18

SOD

activ

ity lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

3

6

9

12

15

18

SOD

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl




0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





























2










2






2










Control


2or MnCl

2for

24 h

1

2

3

4

5

6

7

00 10010


lowast

lowast

Relat

ive m

RNA

expr

essio

n

CdCl2 (120583gL)

(a)

1

2

3

4

5

6

7

00 10 100 1000


lowast

Relat

ive m

RNA

expr

essio

n

MnCl2 (120583gL)

(b)












0

1

2

3

4

0

1

2

3

4

lowast

lowast

0 10010 0 100010010

0 1000100100 10010

HSP70


ld in

crea

se

Fold

incr

ease

120573-Actin

CdCl2 (120583gL)

CdCl2 (120583gL)

MnCl2 (120583gL)

MnCl2 (120583gL)

(a) (b)


2and MnCl






2






2exposure


2(Figure 5(b))









2(Figure 7)


0 10010 0 10010 0 10010

lowast

lowast

1

2

3

4

5

6

7

lowastlowast

lowast

1

2

3

4

5

6

7

1

2

3

4

5

6

7


0 0 0

sod cat gst

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(a)

0 100010010 0 100010010 0 100010010

lowast

0

lowast

lowast

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(b)


0

3

6

9

12

15

18

SOD

activ

ity lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

3

6

9

12

15

18

SOD

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl




0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Control


2or MnCl

2for

24 h

1

2

3

4

5

6

7

00 10010


lowast

lowast

Relat

ive m

RNA

expr

essio

n

CdCl2 (120583gL)

(a)

1

2

3

4

5

6

7

00 10 100 1000


lowast

Relat

ive m

RNA

expr

essio

n

MnCl2 (120583gL)

(b)












0

1

2

3

4

0

1

2

3

4

lowast

lowast

0 10010 0 100010010

0 1000100100 10010

HSP70


ld in

crea

se

Fold

incr

ease

120573-Actin

CdCl2 (120583gL)

CdCl2 (120583gL)

MnCl2 (120583gL)

MnCl2 (120583gL)

(a) (b)


2and MnCl






2






2exposure


2(Figure 5(b))









2(Figure 7)


0 10010 0 10010 0 10010

lowast

lowast

1

2

3

4

5

6

7

lowastlowast

lowast

1

2

3

4

5

6

7

1

2

3

4

5

6

7


0 0 0

sod cat gst

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(a)

0 100010010 0 100010010 0 100010010

lowast

0

lowast

lowast

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(b)


0

3

6

9

12

15

18

SOD

activ

ity lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

3

6

9

12

15

18

SOD

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl




0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

1

2

3

4

0

1

2

3

4

lowast

lowast

0 10010 0 100010010

0 1000100100 10010

HSP70


ld in

crea

se

Fold

incr

ease

120573-Actin

CdCl2 (120583gL)

CdCl2 (120583gL)

MnCl2 (120583gL)

MnCl2 (120583gL)

(a) (b)


2and MnCl






2






2exposure


2(Figure 5(b))









2(Figure 7)


0 10010 0 10010 0 10010

lowast

lowast

1

2

3

4

5

6

7

lowastlowast

lowast

1

2

3

4

5

6

7

1

2

3

4

5

6

7


0 0 0

sod cat gst

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(a)

0 100010010 0 100010010 0 100010010

lowast

0

lowast

lowast

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(b)


0

3

6

9

12

15

18

SOD

activ

ity lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

3

6

9

12

15

18

SOD

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl




0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0 10010 0 10010 0 10010

lowast

lowast

1

2

3

4

5

6

7

lowastlowast

lowast

1

2

3

4

5

6

7

1

2

3

4

5

6

7


0 0 0

sod cat gst

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(a)

0 100010010 0 100010010 0 100010010

lowast

0

lowast

lowast

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

0

1

2

3

4

5

6

7

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n

Relat

ive m

RNA

expr

essio

n


(b)


0

3

6

9

12

15

18

SOD

activ

ity lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

3

6

9

12

15

18

SOD

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl




0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


0

03

06

09

12

15CA

T ac

tivity

lowast

0 10010CdCl2 (120583gL)

(a)

0

03

06

09

12

15

CAT

activ

ity

lowast

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl



0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 10010CdCl2 (120583gL)

(a)

0

100

200

300

400

500

GST

activ

ity

lowast

lowast

0 100010010MnCl2 (120583gL)

(b)


2or 10 100 and 1000 120583gL MnCl











4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





4 Conclusions







Acknowledgments


References























































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






































































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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