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Environmental Pollution 163 (2012) 218e225

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Environmental Pollution

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Role of non-enzymatic antioxidants on the bivalves’ adaptation to environmentalmercury: Organ-specificities and age effect in Scrobicularia plana inhabitinga contaminated lagoon

I. Ahmad a,b,*, I. Mohmood a, J.P. Coelho a, M. Pacheco b, M.A. Santos b, A.C. Duarte a, E. Pereira a

aDepartment of Chemistry & CESAM, University of Aveiro, 3810-193 Aveiro, PortugalbDepartment of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e i n f o

Article history:Received 23 August 2011Received in revised form2 December 2011Accepted 13 December 2011

Keywords:MercuryScrobicularia planaAgeOrgan-specificitiesAdaptationNon-enzymatic antioxidants

* Corresponding author.E-mail address: ahmadr@ua.pt (I. Ahmad).

0269-7491/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.envpol.2011.12.023

a b s t r a c t

This study aimed to investigate the role of non-enzymatic antioxidants on adaptive skills over time in thebivalve Scrobicularia plana environmentally exposed to mercury. Inter-age (2D, 3D, 4D, 5D year old) andorgan-specific (gills, digestive gland) approaches were applied in bivalves collected from moderately andhighly contaminated sites at Ria de Aveiro (Portugal). S. plana’s adaptive skills were dependent on thecontamination extent; under moderate contamination scenario, the intervention of the different anti-oxidants took place harmoniously, evidencing an adjustment capacity increasing with the age. Underhigher contamination degree, S. plana failed to cope with mercury threat, showing an age-dependentdeterioration of the defense abilities. In organ-specific approach, the differences were particularlyevident for thiol-compounds, since only gills displayed the potential to respond to moderate levels byincreasing non-protein thiols and total glutathione. Under high contamination degree, both organs wereunable to increase thiol-compounds, which were compensated by the ascorbic acid elevation.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Mercury’s known threats to the human health resulted ininternational efforts to reduce its usage and release into the aquaticenvironment. However, historically contaminated sediments stillconstitute a source of mercury to the aquatic organisms, and thusrepresenting an environmental risk due to its bioaccumulation andbiomagnification. Among aquatic organisms, infaunal bivalves areparticularly targeted by mercury contamination due to theirinability to successfully regulate metal uptake (Connell and Miller,1984) and filter-feeding nature integrating the metal from bothwater and sediment compartments. Nevertheless, bivalves are notkilled out-rightly, although exposure to mercury can weaken theirimmune system and make them more susceptible to health prob-lems (Ahmad et al., 2011a). This begs the question: what do bivalvesdo to survive in mercury-contaminated environments? It seemsthat these organisms are able to evolve an adaptive capacityto meet the rising challenge of mercury contamination. In thisdirection, it has been observed that antioxidant enzymes wereinsufficient to prevent mercury-induced peroxidative damage in

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Scrobicularia plana (Ahmad et al., 2011b). Besides enzymatic anti-oxidants, non-enzymatic compounds also serve as an importantbiological defense against environmental pro-oxidant conditions.However, literature revealed an insufficient exploration of the roleof non-enzymatic antioxidants in bivalves, not accomplishinga more holistic approach towards the understanding of antioxidantsystem function and regulation in relation to both endogenousand exogenous sources of reactive oxygen species (ROS). Noreport reflects the non-enzymatic antioxidants variations inbivalves inhabiting mercury-contaminated environments, as wellas the evolutionwith age or the specific responsiveness of differenttissues to mobilizing adaptive processes in this context.

The principal toxic effects of mercury involve interactions withanumber of cellular processes including the formation of complexeswith thiol (eSH) groups, which not only regulate the enzymes butregulate also the cellular redox status. The most commonlyobservedmanifestation ofeSHdepletion is its accompanimentwiththe alteration of reduced glutathione (GSH) towards an oxidized(GSSG) state (Hoffman et al., 2002). GSH is the most abundant non-protein thiol (NP-SH) involved in the detoxification of inorganicmercury, methyl mercury and oxy-radicals in aquatic organisms(Canesi et al., 1999). The available literature also revealed enzymesreacting with potentially toxic endogenous compounds and xeno-biotics using GSH (Wendel et al., 1990; Forman et al., 2008). It is

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225 219

known that mercury forms covalent bonds with GSH and a singlemercury ion can bind to and cause irreversible excretion of twoglutathione molecules (Franco et al., 2009). Moreover, it was alsoreported that, in the presence of reducing compounds such asdithiothreitol, mercuryeGSH complexes are not stable, instead itrelease mercury ions, which disturb GSH metabolism and exertscell-damaging effects.

Ascorbic acid (AsA) is a low molecular weight antioxidant thatinteracts directly with the oxidizing radicals by rapid electrontransfer (Patra et al., 2001), protecting cells from ROS-inducedperoxidative damage (Halliwell, 1994). Moreover, the AsA antioxi-dant potential is also attributed to its ability to regenerate othersmall antioxidant molecules, such as a-tocopherol, GSH and b-carotene. Under physiological conditions, ascorbate is oxidized todehydro-ascorbate and then dehydro-ascorbate is predominantlyconverted back to ascorbate by GSH and NADPH-dependentenzymes (e.g., glutaredoxin, thioredoxin) (Park and Levine, 1996;May, 2000). In contrast, the ratio of dehydro-ascorbate to ascorbatemay dramatically increase in cells and tissues exposed to oxidativestress (Haramaki et al., 1998). This may lead to the irreversibleformation of reactive carbonyl intermediates that subsequentlycause modification of proteins (Fayle et al., 2000; Simpson andOrtwerth, 2000; Linster and Van Schaftingen, 2007). In addition,it binds with mercury ions and reduces mercury-induced DNAdamage due to its nucleophilic properties (Rao et al., 2001). Takinginto consideration the lacunae described above, this researchstudied Scrobicularia plana inhabiting a mercury-contaminatedarea (Laranjo basin, Ria de Aveiro e Portugal, regarded as a ‘fieldlaboratory’ due to the presence of a well-defined mercury gradient)to meet the following main goals: 1) to investigate the role of non-enzymatic antioxidants, such as NP-SH, total glutathione (T-GSH)

Fig. 1. (A) Location of Ria de Aveiro (Portugal); (B) Location (-) of sampling sites at Ria de Avsite at Vagueira assumed as reference (R).

and AsA, against mercury exposure; moreover, due to its role inmaintaining the glutathione redox status, the enzyme glutathionereductase (GR) was also evaluated; 2) to evaluate age-relatedvariations on the previous non-enzymatic antioxidants in orderto clarify the evolution of adaptive skills over time; and 3) to assessorgan-specificities by analyzing gills and digestive gland responses,as organs differing on their defensive capacity and anatomic loca-tion, which determines exposure routes, accumulation propensityand pro-oxidant challenge extent.

2. Materials and methods

2.1. Study area and sampling

Laranjo basin is the most mercury-contaminated area in the Ria de Aveiro(Portugal), a coastal lagoon, which received chlor-alkali plant discharges continu-ously for five decades, resulting in the generation of a mercury contaminationgradient. Though in recent past (approximately 15 years ago) this industry hasstopped the effluent release, high mercury levels are still present in the sediments(Ahmad et al., 2011b) and its progressive re-suspension, mainly during the periodsof stronger tidal currents, are responsible for metal exportation and increasedbioavailability (Pereira et al., 2009).

The sampling was carried out in July 2008 in three sites: two sites at Laranjobasin, termed as M (moderately contaminated) and H (highly contaminated), anda third site as reference (R), at Vagueira, considered unpolluted including in terms ofmercury (Fig. 1). This site belongs to an area, far from the main mercury pollutionsource (around 30 km), where neither industry in its surrounding, nor urban effluentoutlets of noteworthy importance are present (Duarte et al., 2007; Pereira et al.,2009). An incremental trend of mercury associated with sediment and suspendedparticulate matter was observed with the proximity to mercury source. In light ofsediment quality guidelines and the value for “Effects Range-Low” (0.15 mg kg�1)proposed by Long et al. (1995), the sitesM and H hadmercury concentrations 27 and132 times higher than the proposed level (Ahmad et al., 2011b). At each site, Scro-bicularia plana (Peppery furrow shell) specimens were collected during low tideconditions by digging out to a depth of 30 cm on the mudflats and washed through

eiro: moderately (M) and highly (H) mercury-contaminated sites at Laranjo basin, and a

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225220

1 mm mesh bags. The collected animals were divided into age groups, classifiedaccording to the shell yearly growth increments as proposed by Verdelhos et al.(2005): 2þ, 3þ, 4þ and 5þ year old clams.

The present study encompassed two components: (i) inter-age approachewhereall the parameters were assessed in the whole soft tissues, addressing inter-agecomparison and thus, using different age classes (2þ to 5þ year old clams);(ii) organ-specific approach e where all the parameters were assessed in specificorgans, viz. gills and digestive gland, in 4þ year clam (the choice of this class was basedon animals availability and the body mass in order to provide enough material toanalyze). Accordingly, ten animals destined for inter-age approachwere dissected andthe whole soft tissues immediately frozen in liquid nitrogen and stored at �80 �C forantioxidants assessment. Animals devoted to the organ-specific approach alsoincluded ten specimens. Animals were dissected and gills and digestive gland werecollected for antioxidants assessment. The methodology in terms of animals handlingand sampling processingwas the same as previously described for inter-age approach.

2.2. Biochemical analyses

In laboratory, samples (whole soft tissues or organs) were homogenized, usinga PottereElvehjem homogenizer, in chilled sodium phosphate buffer (0.1 M,pH ¼ 7.4) (1 g of tissue/10 mL buffer). This homogenate was then divided in twoaliquots for T-GSH quantification as well as for post-mitochondrial supernatant(PMS) preparation. The PMS preparation was accomplished by centrifugation ina refrigerated ultracentrifuge (Optima TL, Beckman) at 13,400 g for 20 min at 4 �C.Aliquots of PMS were stored at �80 �C until analyses.

2.2.1. Total non-protein thiols (NP-SH) contentNP-SH was determined by the method of Sedlak and Lindsay (1968) as adopted

by Parvez et al. (2003). PMS (0.3 mL) was precipitated with 0.3 mL of sulfosalicylicacid (5%). The samples were kept at 4 �C for 1 h and then subjected to centrifugationat 13,400 g for 15 min at 4 �C. The assay mixture contained 0.5 mL of deproteinatedsupernatant, 2.3 mL of sodium phosphate buffer (0.1 M, pH 7.4) and 0.2 mL 2,5,dithiobis-tetranitrobenzoic acid (DTNB) (stock ¼ 10 mM in 0.1 M sodium phosphatebuffer, pH 7.4) in a total volume of 3 mL. The optical density of reaction product wasread immediately at 412 nm on a spectrophotometer (UV/VIS, Spectramax 384). Theresults were expressed as mmol 5-thio-2-nitrobenzoic acid (TNB) formed/g tissueusing a molar extinction coefficient of 13.6 � 103 M�1 cm�1.

2.2.2. Total glutathione (T-GSH) contentT-GSH was determined in a different homogenate aliquot in relation to NP-SH.

Protein content in the tissue homogenate was precipitated and centrifuged asdescribed above, and the resulting supernatant was used for T-GSH determination,adopting the enzymatic recyclingmethod using GR, whereby the sulfhydryl group ofT-GSH reacts with DTNB producing a yellow colored TNB. The rate of TNB productionis directly proportional to this recycling reaction, which is in turn directly propor-tional to the concentration of T-GSH in the sample (Tietze, 1969; Baker et al., 1990).Formation of TNB was measured by spectrophotometry at 412 nm at 25 �C. It shouldbe noted that oxidized glutathione is converted to reduced glutathione by GR in thissystem, which consequently measures T-GSH. The results were expressed as mmolTNB formed/min/g tissue using amolar extinction coefficient of 14.1�103M�1 cm�1.

2.2.3. Ascorbic acid (AsA) contentAsA content was determined in PMS fraction, using AsA assay kit (Product n� ab

65346, Abcam plc 330 Cambridge Science Park, Cambridge CB4 0FL, UK). In thisassay, the proprietary catalyst oxidizes AsA to produce a product that interacts withthe AsA-probe and generates color. The absorbance was recorded at 570 nm usinga standard 96-well plate reader (UV/VIS, Spectramax 384). Results were expressedas mmol AsA/g tissue.

2.2.4. Glutathione reductase (GR) activity measurementGR activity was assayed by the method of Cribb et al. (1989) with some modi-

fications. Briefly, the assay mixture contained 0.025 mL of PMS fraction and0.925 mL of NADPH (0.2 mM), GSSG (1 mM) and DTPA (diethylenetriaminepenta-acetic acid; 0.5 mM). The enzyme activity was quantified at 25 �C by measuring thedisappearance of NADPH at 340 nm during 3 min. The enzyme activity was calcu-lated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficientof 6.22 � 103 M�1 cm�1.

Total protein contents were determined according to the Biuret method (Gornallet al., 1949), using bovine serum albumin as a standard.

2.3. Data analysis

SPSS (PASW statistics 18) for Windows was used for statistical analysis of data.Descriptive statistics was initially performed on the data and homogeneity of variancewas tested. ANOVA followed by the Tukey’s test was performed in order to compareresults between sites (within the same age class) or between age classes (within thesame sites), while correlations between variables were tested through Pearson’scorrelation. Interactions between subject factors (age � site) were analyzed usingunivariate analysis of variance. All the results significance was ascertained at p< 0.05.

3. Results

Hydrological parameters (Temperature: 23e28 �C; pH: 7.8e8.4;dissolved oxygen: 5.4e8.4; salinity: 22e27&) in the surveyed siteswere on acceptable level considering the criteria given in APHA(Clesceri et al., 1998) as well as bivalves’ demands.

3.1. Inter-age approach

Concerning inter-site comparisons (Fig. 2), 2þ year clams depictedsignificantly higher NP-SH content at M and AsA at H, whereasa lower GR activity was observed at H in comparison to thosecollected at R. In addition, comparing toM, significantly lower NP-SHcontent andhigherAsAcontentwere observedatH. Inwhat concernsto 3þ year clams, it was registered a similar inter-site responsepattern in terms of NP-SH as described for 2þ year clams; however, interms of T-GSH, an increase at M and Hwas perceptible, whereas GRshowed a significant decrease at M (always in relation to R). Asignificantly lower T-GSH at H was also perceptible in comparison toM. In relation to 4þ year clams, it was noticeable a NP-SH increase atM, as well as a T-GSH and AsA increase at both M and H sites,compared to R. This class also showed lower GR activity at M andH sites. At H, a significant decrease in NP-SH was observed incomparison to M. Finally, 5þ year clams showed significant decreasein NP-SH at M and H, as well as in T-GSH and GR activity at H, incomparison to R. A higher GR activity was observed at M incomparison to R. In addition, comparing to M, significantly higherNP-SH content and lower T-GSH content were observed in 5þ yearclams.

Regarding inter-age comparison of antioxidant responses (withinthe same site), no significant differenceswere observed at R for anyofthe parameters studied (Fig. 2). At M site, a significant NP-SHdecrease was observed in 5þ year clams in comparison to all theother classes, whereas for T-GSH content, a significant increase wasobserved in 3þ year clams compared to all the other classes. Con-cerning AsA content at M, a significant increase was observed in 4þ

and 5þ year clams comparing with 2þ and 3þ year clams. On theperspective of GR, a decreased activity in 3þ, 4þ, and 5þ year clamswas observed when compared to 2þ year clams. Significantlyincreased activity was also observed when 5þ year clams werecompared with 3þ year clams.

At H site, significant inter-age differences were only observedfor T-GSH content, which showed a lower value in 5þ year clamswhen compared to 2þ year clams, and for AsA content showinga higher value in 3þ year clams when compared to 2þ yearclams.

Pearson’s analysis within each age class (Fig. 3) revealed positivecorrelations between NP-SH and T-GSH in 3þ year class, betweenAsA and GR in 4þ year class, as well as between T-GSH and AsA andNP-SH and GR in 5þ year clams. As per the age and site interactions,all the studied parameters (except GR) reflected significant inter-actions (Table 1).

3.2. Organ-specific approach

Concerning gills non-enzymatic antioxidants (Fig. 4), significantlyhigher NP-SH and T-GSH contents aswell as GR activitywas detectedat M, whereas at H, significantly lower NP-SH and higher AsAcontent was observed (always vs. R). In digestive gland, a signifi-cantly lower NP-SH and higher AsA content was observed at both thecontaminated sites, comparing to R. Moreover, significantly higherAsA content was perceptible at H in comparison to M site. Pearson’scoefficients revealed a significant negative correlation (r ¼ 0.566)between NP-SH and AsA among all the studied parameters indigestive gland.

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Fig. 2. Values represent the means (�standard error) of non-protein thiols (NP-SH), total glutathione (T-GSH), ascorbic acid (AsA) and glutathione reductase (GR) measured in thewhole body of Scrobicularia plana of different age classes (2þ, 3þ, 4þ and 5þ year old) collected at reference (R), moderately (M) and highly (H) mercury-contaminated sites in the Riade Aveiro. Statistically significant differences (p < 0.05) are marked by letters (differences between sites, within the same class) and numbers (differences between classes, withinthe same site): a vs. R, b vs. M; 2 vs. 2þ year; 3 vs. 3þ year; 4 vs. 4þ year; 5 vs. 5þ year.

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225 221

4. Discussion

In a sequence of our previous studies (Ahmad et al., 2011a,b),the current study constitutes a further step in order to achieve theunderstanding of bivalves’ protection responses to environmentalmercury exposure. Non-enzymatic antioxidants protection seemsto be an important indirect mechanism against oxidative stressinduced by mercury since it is a redox-inactive metal and does notundergo redox cycling (Stohs and Bagchi, 1995). On the otherhand, organisms evolve important adaptive changes in responseto sulfhydryl-reactive metals such as mercury. Our previousresults demonstrated that bivalve environmental exposure tomercury induces immunotoxicity (Ahmad et al., 2011a), alsorevealing that whole-body analyses can be particularly limitingwhen enzymatic antioxidants are considered (Ahmad et al.,2011b). The whole-body accumulation as total mercury changedaccording to the environmental gradient for all age classes, whilein terms of organic mercury, only 5D year animals showedincreased accumulation with increased environmental mercurylevel (Ahmad et al., 2011b). Moreover, based on the data formercury in water, sediment and whole soft tissues, it was clearthat the study area is still under recovery state. Thus, keeping inview the previous scenario, this accompanying study addressedthe role of non-enzymatic antioxidants protection againstmercury, using two approaches viz. inter-age and organ-specific,and the responses have been related with previously publishedbioaccumulation data (Ahmad et al., 2011b).

4.1. Inter-age approach

In the present study, clam’s responses displayed a divergentpattern of variation depending on the degree of contamination.Under moderate contamination conditions (site M), the oldest clams(5D year) failed to respond to mercury accumulation through anelevation of bothNP-SH and T-GSH contents. The age-related patternof GR variation fits with the T-GSH response, since the highest T-GSHlevel was observed for 3þ year clams coincidentlywith the lowest GRactivity level (also significantly lower than R) and the absence ofT-GSH elevation observed for 5D year clams coincided temporallywith the only significant increase of GR activity (vs. R). T-GSHdepletion may be attributed to the fact that mercury generateshighly toxic hydroxyl radicals from the breakdown of hydrogenperoxide, which further deplete T-GSH stores (Lee et al., 2001).Elevated GR activity was observed in 5D year clams in response tomercury accumulation reflecting increased glutathione recycling(replenishing GSH in order to avoid its depletion), suggesting thatthe ratio GSSG/GSH has been increased. GR plays a major role inglutathione peroxidase (GPX) and glutathione S-transferase (GST)reactions as an adjunct in the control of peroxides and free radicals(Bompart et al., 1990), maintaining the proper GSH redox status.Moreover, if we compare the current GR responses at site M withthose of other enzymes such as GPX and GST, as well as catalase(CAT), reported in a previous study (Ahmad et al., 2011b) to the samesampling site, it should be highlighted the occurrence of distinctresponse patterns. Thus, the only age class to showactivity reduction

r = 1.00

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Fig. 3. Pearson’s correlation coefficients (r) determined within each age class, between the different parameters measured in the whole body of Scrobicularia plana (p < 0.05).Non-protein thiols e NP-SH; Total glutathione e T-GSH; ascorbic acid e AsA; glutathione reductase e GR. Non significant correlations are not presented.

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225222

for GPX and GST was 2þ, which didn’t show that susceptibility forGR. Moreover, in GR, that vulnerability towards inhibition seemed tohave been transferred to classes 3þ and 4þ, which showed no activityreduction in the cases of CAT, GPX and GST. As a further evidence ofdissimilar modulations principles, GR was currently induced in 5þ

year clams, while CAT, GPX and GST didn’t reveal any tendency to beinduced in this class (Ahmad et al., 2011b).

In turn, AsA demonstrated an opposite age-related variationpattern in relation to NP-SH and T-GSH (higher increments in 4D and5D year clams compared to 2D and 3D year clams atM), pointing outa compensatory role of this low molecular weight antioxidant.Overall, this integrated response profile atM site, and particularly theresponses registered for 5D year clams, should be regarded as anadjustment phenomenon occurring over time, rather than as anorganism limitation to react defensively tomercury body burden andsubsequent (potential) damaging actions. This explanation is sup-ported by the maintenance of similar total mercury accumulation

Table 1Univariate analysis of variance testing the effect of age, site and interaction (age� site)on the levels of non-enzymatic antioxidants in Scrobicularia plana. The F and p valuesare given for each variable. Non-protein thiols e NP-SH; total glutathione e T-GSH;ascorbic acid e AsA and glutathione reductase e GR; ns e non significant.

Dependentvariables

Age Site Age � Site

F p F p F p

NP-SH 0.747 ns 10.99 <0.05 4.980 <0.05T-GSH 2.673 <0.05 5.444 <0.05 6.172 <0.05AsA 2.898 <0.05 5.268 <0.05 2.995 <0.05GR 2.166 ns 2.528 ns 0.757 ns

from the 3þ to 5þ year and the absence of lipid peroxidation (LPO)increases for 4D and 5D year clams (Ahmad et al., 2011b). Despite theincapacity to completely avoid LPO increase (namely in 2þ and 3þ

classes) (Ahmad et al., 2011b), the defenses presently assessed seemto play a compensatory role in relation to the general failure of theenzymatic antioxidants previously measured (Ahmad et al., 2011b).

Under a high contamination degree, the response profiles werecompletely different. As a premise to discuss the results from siteH, itshould be noticed that total mercury accumulationwas substantiallyhigher in 4D and 5D year clams in relation to the younger classes(Ahmad et al., 2011b). S. plana captured at site H displayed, for all theage classes, the incapacity to respond to mercury accumulation riseby increasing the bulk of NP-SH. Similarly, 2D and 5D year clamsfailed to increase T-GSH content. Furthermore, the significant T-GSHdecrease measured in 5D year clams at site H, relatively to R, isindicative of an imbalance in redox status and increased risk towardsmercury pro-oxidant action. This T-GSH lowering effect can beattributed to the formation of mercuryeGSH complexes and subse-quent metal elimination and/or to an obstruction of GSH newsynthesis as a result of an inhibitory action on the enzymes involvedin that process (gamma-glutamylcysteine synthetase and GSHsynthetase). In contrast with what was described for site M, GR wasunable to play its role in maintaining the glutathione redox status in2D, 4D and 5D year clams, as demonstrated by the significant activitydecreases measured in relation to site R. This could mean a vulnera-bility towards a decrease in the ratio GSH/GSSG by the action ofmercury. In 5D year clams from site H, the apparent limitation of GRaction was not accompanied by a similar response profile in whatconcerns to CAT, GST and GPX, as reported by Ahmad et al. (2011b).

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Fig. 4. Values represent the means (�standard error) of non-protein thiols (NP-SH), total glutathione (T-GSH), ascorbic acid (AsA) and glutathione reductase (GR) measured in gillsand digestive gland of Scrobicularia plana (4þ year old specimens) collected at reference (R), moderately (M) and highly (H) mercury-contaminated sites in the Ria de Aveiro.The significant differences between sites are: a vs. R, b vs. M.

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225 223

Since induction of antioxidants represents a cellular defense mech-anism to counteract toxicity of ROS, they have been extensively usedin several field studies to assess the extent of pollution in coastalwaters (Goksoyr, 1995). Apart from major antioxidants assessed inthe current study, there are certainly other antioxidant enzymes,which may clarify the complete scenario of the oxidative stressdefense during the life span of animal in response to mercury accu-mulation. These enzymes serve as a backup function by replenishingGSH from glutathione disulfide (GSSG) by the enzyme GR and thereducing equivalent is provided by the enzyme G6PD (Rodriguez-Ariza et al., 1991). In addition to the enzymes, data on singledefenses can be integrated with the total oxyradical scavengingcapacity (TOSC) assay which measures the capability of the wholeantioxidant systemtoneutralize variousoxy-radicals, quantifying theresistance of a biological tissue against contaminants (Regoli et al.,2004). Concerning AsA response, it should be highlighted that 5D

year clamswere the only age class showing the inaptitude to respondto mercury load elevation. Again, in contrast to site M, a coordinatedaction of AsA and NP-SH as well as T-GSH was not apparent. In thesame direction, the general loss of non-enzymatic antioxidantsproficiency detected in the oldest animals (5D year clams) should beinterpreted as a deterioration of the defense system abilities, whichcan be on the basis of the LPO increase concomitantly observed in ourprevious study (Ahmad et al., 2011b). In line with what wasstated above for the moderate contamination scenario, underhigher contamination degree a balance enzymatic/non-enzymatic

antioxidants seems to be attempted by the organisms, namely in5D year clams, though in this case therewere enzymatic antioxidants(CAT, GPX and GST) (Ahmad et al., 2011b) to compensate thenon-enzymatic antioxidants failure.

4.2. Organ-specific approach

Looking first to gills responses, it was evident that this organreacted to the exposure to moderate levels of mercury (site M) byincreasing -SH containing compounds (NP-SH and T-GSH) and therelated enzyme (GR), coping with the slight increase in mercuryaccumulation e only the organic form increased significantly(Ahmad et al., 2011b)ewithout the intervention of AsA. The bindinganddissociation ofmercury-SH complexes are believed to control themovement of mercury and its toxic effects in aquatic animals(Clarkson, 2002). These gills responses in terms of non-enzymaticantioxidants, together with catalase induction (Ahmad et al.,2011b), were able to impair the appearance of LPO in this organ(Ahmad et al., 2011b). Differently, at site H, gills were unable torespond to the accentuated increase on mercury accumulation, bothtotal and organic forms (Ahmad et al., 2011b), through NP-SH andT-GSH, which was compensated by the elevation of AsA content. Inthis case, the antioxidant systemwas impotent to avoid peroxidativedamage (Ahmad et al., 2011b).

Concerning the digestive gland, probably as a result of thehigher levels of mercury accumulation (mainly in the inorganic

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225224

form) as compared to gills (Ahmad et al., 2011b), it was observed anexhaustion of NP-SH levels in both M and H sites accompanied bythe incapacity of T-GSH to respond. Once more, AsA seemed to playa compensatory role, showing the ability to respond proportionallyto the exposure extent. This apparent complementary action of AsAand thiols is supported by the significant negative correlationdetected between AsA and NP-SH contents. Surprisingly, LPOincrease was only observed at site M (Ahmad et al., 2011b). Thispattern of damage induction suggests the preponderant protectiverole of AsA, since in the presence of a great increment of this lowmolecular weight antioxidant (also significantly higher at H inrelation to M), lipid peroxidation was avoided, even in the absenceof any enzymatic antioxidant action (Ahmad et al., 2011b).Comparing the performance, in digestive gland, of non-enzymaticantioxidants currently assessed and the enzymatic antioxidantspreviously evaluated (Ahmad et al., 2011b), it is noteworthy that theformers were clearly more responsive, which can be related toa higher vulnerability of enzymes towards mercury-induced toxicactions.

Taking both gills and digestive glands responses (and even thewhole-body responses discussed in the inter-age approach), it wasnotorious that, among the studied non-enzymatic antioxidants,eSHcompounds are a first line of defense, but along with a mercurytissue load elevation (occurring with the increased external levels ofexposure or with the increased age) their role tend to be played byAsA.

It is also interesting to compare both studied organs in terms oftheir basal levels of antioxidants (measured at the reference site),since it could be important on determining the need to or thepotential to respond (with rising levels) to mercury exposure. Inthis perspective, it was evident that digestive gland displayedsubstantially higher levels of T-GSH (3 times higher) and GR(6 times higher) than gills.

Comparing organ-specific with whole animal responses, it wasobvious that gills followed a general pattern more similar to thatone displayed by whole soft tissues.

5. Conclusions

Overall, current results demonstrated the importance of non-enzymatic antioxidants on protecting S. plana from mercurypro-oxidant action, under environmentally realistic conditions.

In elucidating the age-related variations on non-enzymaticantioxidant responses, it was demonstrated that the evolution ofthe adaptive skills of S. plana over time depends on the contami-nation extent. Hence, under a moderate contamination scenario(site M), the intervention of the different antioxidants (measured inwhole-body) took place harmoniously, evidencing an adjustmentcapacity increasing with the age. In opposition, under a highercontamination degree (site H) S. plana failed to efficiently copewithmercury threat, showing an age-dependent deterioration of thenon-enzymatic defense abilities.

Though both studied organs reflected a pro-oxidant chal-lenge posed by mercury, organ-specific responses were evident.The differences were particularly evident for eSH compounds,since only gills displayed the potential to respond to moderatelevels of mercury by increasing NP-SH and T-GSH contents(as well as GR activity). Under high contamination degree, thedifferences between gills and digestive gland were mitigated, asboth organs were unable to increase eSH compounds, whichwas compensated by the elevation of AsA content. AsA showedto be the assessed antioxidant that better reflected the externallevels of exposure, which seems to be related with a lesservulnerability to limitations imposed by high levels of mercuryaccumulation.

Acknowledgments

Thefinancial support provided by the Portuguese FCT (Foundationfor Science and Technology) to CESAM(Centre for Environmental andMarine Studies) is gratefully acknowledged.

References

Ahmad, I., Coelho, J.P., Mohmood, I., Pacheco, M., Santos, M.A., Duarte, A.C.,Pereira, E., 2011a. Immunosuppression in the infaunal bivalve Scrobiculariaplana environmentally exposed to mercury and association with its accumu-lation. Chemosphere 82, 1541e1546.

Ahmad, I., Mohmood, I., Mieiro, C.L., Coelho, J.P., Pacheco, M., Santos, M.A.,Duarte, A.C., Pereira, E., 2011b. Lipid peroxidation versus antioxidant modula-tion in the bivalve Scrobicularia plana in response to environmental mercu-ryeorgan specificities and age effect. Original research article. AquaticToxicology 103, 150e158.

Baker, M.A., Cerniglia, G.J., Zaman, A., 1990. Microtiter plate assay for the measure-ment of glutathione and glutathione disulfide in large numbers of biologicalsamples. Analytical Biochemistry 190, 360e365.

Bompart, G.J., Prevot, D.S., Bascands, J.-L., 1990. Rapid automated analysis ofglutathione reductase, peroxidase, and S-transferase activity: application tocisplatin-induced toxicity. Clinical Biochemistry 23, 501e504.

Canesi, L., Viarengo, A., Leonzio, C., Filippelli, M., Gallo, G., 1999. Heavy metals andglutathione metabolism in mussel tissues. Aquatic Toxicology 46, 67e76.

Clarkson, T.W., 2002. The three modern faces of mercury. Environmental HealthPerspectives 110, 11e23.

Clesceri, L.S., Greenberg, A.E., Eaton, A.D., 1998. American Public Health Association(APHA), Standard Methods for the Examination of Water and Wastewater,20th ed. APHA, D.C. Washington.

Connell, D.W., Miller, G.J., 1984. Chemistry and Ecotoxicology of Pollution.John Wiley & Sons, New York.

Cribb, A.E., Leeder, J.S., Spielberg, S.P., 1989. Use of a microplate reader in an assay ofglutathione reductase using 5,50-dithiobis (2-nitrobenzoic acid). AnalyticalBiochemistry 183, 195e196.

Duarte, A., Rodrigues, S., Pato, P., Coelho, C., Pereira, M.E., 2007. A review on studiesof mercury contamination in the coastal lagoon ‘Ria de Aveiro’ Portugal.La Houille Blanche 4, 35e39.

Fayle, S.E., Gerrard, J.A., Simmons, L., Meade, S.J., Reid, E.A., Johnston, A.C., 2000.Crosslinkage of proteins by dehydroascorbic acid and its degradation products.Food Chemistry 70, 193e198.

Forman, H.J., Zhang, H., Rinna, A., 2008. Glutathione: overview of its protectiveroles, measurement, and biosynthesis. Molecular Aspects of Medicine 30, 1e12.

Franco, R., Sánchez-Olea, R., Reyes-Reyes, E.M., Panayiotidis, M.I., 2009. Environ-mental toxicity, oxidative stress and apoptosis: ménage à trois. MutationResearch 674, 3e22.

Goksoyr, S.G., 1995. Use of cytochrome P4501A (CYP1A) in fish as a biomarker ofaquatic pollution. Archives of Toxicology 17, 80e95.

Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum proteins bymeans of the Biuret reaction. Journal of Biological Chemistry 177, 751e766.

Halliwell, B., 1994. Free radicals, antioxidants, and human disease: curiosity, causeand consequence? Lancet 344, 721e724.

Haramaki, N., Stewart, D.B., Aggarwal, S., Ikeda, H., Reznick, A.Z., Packer, L., 1998.Networking antioxidants in the isolated rat heart are selectively depleted byischemiaereperfusion. Free Radical Biology and Medicine 25, 329e339.

Hoffman, D.J., Marn, C.M., Marois, K.C., Sproul, E., Dunne, M., Skorupa, J.P., 2002.Sublethal effects in avocet and stilt hatchlings from selenium-contaminatedsites. Environmental Toxicology and Chemistry 21, 561e566.

Lee, Y.W., Ha, M.S., Kim, Y.K., 2001. Role of reactive oxygen species and glutathionein inorganic mercury induced injury in human glioma cells. NeurochemicalResearch 26, 1187e1193.

Linster, C.L., Van Schaftingen, E., 2007. Vitamin C: biosynthesis, recycling anddegradation in mammals. The FEBS Journal 274, 1e22.

Long, E.R., MacDonald, D.D., Smith, S.L., Calder, F.D., 1995. Incidence of adversebiological effects within ranges of chemical concentrations in marine andestuarine sediments. Journal of Environmental Management 19, 81e97.

May, J.M., 2000. How does ascorbic acid prevent endothelial dysfunction?Free Radical Biology and Medicine 28, 1421e1429.

Park, J.B., Levine, M., 1996. Purification, cloning and expression of dehydroascorbicacid-reducing activity from human neutrophils: identification as glutaredoxin.Biochemistry Journal 315, 931e938.

Parvez, S., Sayeed, I., Pandey, S., Ahmad, A., Hafeez, B., Haque, R., Ahmad, I.,Raisuddin, S., 2003. Regulatory role of copper on non-enzymatic antioxidants infreshwater fish Channa punctatus (Bloch.). Biological Trace Element Research 93,237e248.

Patra, R.C., Swarup, D., Dwivedi, S.K., 2001. Antioxidant effects of a tocopherol,ascorbic acid and L-methionine on lead induced oxidative stress to the liver,kidney and brain in rats. Toxicology 162, 81e88.

Pereira, M.E., Lillebø, A.I., Pato, P., Válega, M., Coelho, J.P., Lopes, C., Rodrigues, S.,Cachada, A., Otero, M., Pardal, M.A., Duarte, A.C., 2009. Mercury pollution in Riade Aveiro (Portugal): a review of the system assessment. EnvironmentalMonitoring Assessment 155, 39e49.

I. Ahmad et al. / Environmental Pollution 163 (2012) 218e225 225

Rao, M.V., Chinoy, N.J., Suthar, M.B., Rajvanshi, M.I., 2001. Role of ascorbic acid onmercuric chloride-induced genotoxicity in human blood cultures. Toxicologyin Vitro 15, 649e654.

Regoli, F., Frenzilli, G., Bocchetti, R., Annarumma, F., Scarcelli, V., Fattorini, D., Nigro,M., 2004. Time-course variations of oxyradical metabolism, DNA integrity andlysosomal stability in mussels, Mytilus galloprovincialis, during a field trans-location experiment. 68, 167e178.

Rodriguez-Ariza, A., Dorado, G., Peinado, J., Pueyo, C., Lopez-Barea, J.,1991. Biochemicaleffects of environmental pollution in fishes from Spanish South-Atlantic littoral.Biochemical Society Transactions 19, 301S.

Sedlak, J., Lindsay, H.R., 1968. Estimation of total, protein-bound and nonproteinsulfhydryl groups in tissues with Ellman’s reagent. Analytical Biochemistry 25,192e205.

Simpson, G.L., Ortwerth, B.J., 2000. The non-oxidative degradation of ascorbic acidat physiological conditions. Biochimica et Biophysica Acta 1501, 12e24.

Stohs, S., Bagchi, J., 1995. Oxidative mechanisms in the toxicity of metals ions.Free Radical Biology and Medicine 2, 321e336.

Tietze, F., 1969. Enzymatic method for quantitative determination of nanogramamounts of total and oxidized glutathione. Analytical Biochemistry 27,502e522.

Verdelhos, T., Neto, J.M., Marques, J.C., Pardal, M.A., 2005. The effect of eutrophi-cation abatement on the bivalve Scrobicularia plana. Estuarine Coastal and ShelfScience 63, 261e268.

Wendel, A., Tiegs, G., Werner, C., 1990. Manipulation of liver glutathione statusdadouble-edged sword. In: Viña, J. (Ed.), Glutathione: Metabolism and Physio-logical Functions. CRC Press, Boca Raton, Florida, pp. 21e28.