gy, Part C 143 (2006) 187–195www.elsevier.com/locate/cbpc

Comparative Biochemistry and Physiolo

Biochemical biomarkers in gills of mangrove oyster Crassostrea rhizophoraefrom three Brazilian estuaries

Juliano Zanette a, José Maria Monserrat b, Adalto Bianchini b,⁎

a Programa de Pós-Graduação em Oceanografia Biológica, Fundação Universidade Federal do Rio Grande, 96201-900, Campus Carreiros,Rio Grande, RS, Brazil

b Departamento de Ciências Fisiológicas, Fundação Universidade Federal do Rio Grande, Departamento de Ciências Fisiológicas, 96201-900,Campus Carreiros–Av. Itália km 8, Rio Grande, RS, Brazil

Received 17 July 2005; received in revised form 29 January 2006; accepted 8 February 2006Available online 14 February 2006

Abstract

Responses of biochemical biomarkers were evaluated in gills of immature adult mangrove oyster Crassostrea rhizophorae collected in threeestuarine regions along the Brazilian coast. In each region, ten oysters were collected in one reference site (R) located far from pollution sources,and in two polluted sites (P-I and P-II sites) located in another water body with similar characteristics. P-I site is located close to recognizedpollution sources while P-II site is in the same water body, but far from pollution sources. At the Paranaguá Bay (Southern Brazil), polluted sitesreceive domestic, harbor and phosphate fertilizer plant discharges. High lipid peroxides (LPO) content was observed in winter oysters from the P-Isite. In summer, higher catalase activity was observed in these oysters. In the Piraquê region (Southeastern Brazil), polluted sites receive domesticand agricultural effluents. Lower total oxyradical scavenging capacity (TOSC) towards peroxyradicals was observed in summer oysters from bothP-I and P-II sites. In the Itamaracá region (Northeastern Brazil), polluted sites receive paper mill and caustic soda and chlorine factories effluents.Increased glutathione S-transferase (GST) activity was observed in oysters from the P-I site in both summer and winter. At Paranaguá Bay (higherlatitude), no seasonal differences were observed in oysters from the R site, suggesting that temperature was not an important factor influencingbiomarkers levels. Lower GST activity was observed in oysters from the R site of the Itamaracá Bay (lower latitude) in winter and summer. Takentogether, data obtained point to responses of biomarkers in oysters from polluted sites of the three estuarine regions analyzed, indicating the needfor future monitoring of the biological effects of contaminants in these environments. They also point to the relevance to consider both season andlatitude as factors influencing biomarker responses in environmental contamination monitoring programs.© 2006 Elsevier Inc. All rights reserved.

Keywords: Biomarkers; Crassostrea rhizophorae; Estuary; Mangrove oyster; Oxidative stress; Pollution monitoring; Seasonality; TOSC

1. Introduction

The mangrove oyster Crassostrea rhizophorae (Bivalvia:Ostreidae) is an euryhaline species inhabiting mangroves andestuaries with a wide distribution from Southern Brazil to theCaribbean (Boffi, 1979). Like several other mollusk bivalves,C. rhizophorae has important characteristics as a useful sentinelspecies in pollution biomonitoring (Wallner-Kersanach et al.,2000; Rebelo et al., 2003; Silva et al., 2003).

⁎ Corresponding author. Tel.: +55 53 233 6853; fax: +55 53 2336850.E-mail address: adalto@octopus.furg.br (A. Bianchini).

1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpc.2006.02.001

Biochemical responses in aquatic organisms have beenemployed in several monitoring programs to characterizeanthropogenic pollution (Burgeot et al., 1996; Cajaraville etal., 2000). Biochemical biomarkers can be broadly classifiedinto two categories: one that measures only exposure to apollutant and another that measures both exposure and toxiceffects (Walker, 1998). Some biomarkers are involved in thebiotransformation of organic xenobiotics to more polarmolecules that are more easily released from the cell.Glutathione S-transferases (GSTs) are the major phase II-relatedenzymes, conjugating electrophilic compounds with glutathione(GSH). GSTs have evolved for cellular protection against arange of xenobiotics and oxidative metabolic by-products.

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There are at least eight major classes of isoforms of this enzyme(Boutet et al., 2004).

Antioxidant defenses are studied to denote pollution, sincegeneration of reactive oxygen species (ROS) could be increasedby the presence of some contaminants such as metals. In addition,oxidation of organic xenobiotics (e.g., hydrocarbons) generatesthe superoxide radical (O2

−) via redox cycle during phase I ofbiotransformation processes (Kappus, 1986). Antioxidantdefenses include both enzymatic (e.g., catalase, glutathioneperoxidase and superoxide dismutase) and non-enzymatic ones(e.g., reduced glutathione, ascorbic acid, uric acid, vitamin E andβ carotene) (Storey, 1996). In this way, measurement of a singleantioxidant is not enough to define a complete antioxidantdefense under experimental or natural conditions. In the lastyears, several methods to measure the whole antioxidant capacityof an organism have been developed. The “total oxyradicalscavenging capacity” assay (TOSC) was developed by Winstonet al. (1998) to estimate the total scavenging capacity towardsdifferent oxyradicals (e.g., peroxyradical, oxyradical and perox-ynitrite radical) in biological samples.

When pollution causes an imbalance of the cellular redox-status, damages in biomolecules such as DNA, lipids andproteins, and mutagenic and carcinogenic processes can occur(Sies and Stahl, 1992). Increased lipid peroxide (LPO) is one ofthe major contributors to the loss of cell function in oxidativestress situations (Hermes-Lima et al., 1995). In this way, LPOdetermination has been employed to denote an oxidative stresssituation in invertebrates (Monserrat et al., 2003).

Other useful biochemical biomarkers are metallothioneins,which point to metal contamination in the aquatic environment.Metallothioneins or metallothionein-like proteins (MTs) are lowmolecular weight proteins, rich in cysteine. They bind metalsbringing them to a non-toxic form, thus reducing theirdeleterious effects (Viarengo et al., 1997).

Several studies have investigated the seasonal variations onsome biochemical biomarker levels, linking them to seasonalchanges in physicochemical parameters like temperature, foodavailability and an animal's seasonal endogenous cycles such asreproduction and growth (Sheehan and Power, 1999; Niyogi etal., 2001; Regoli et al., 2002).

In light of the above, the aim of the present study was toevaluate the response of several biochemical biomarkers in themangrove oyster C. rhizophorae collected at polluted and non-polluted sites from three estuaries along the Brazilian coast.Possible changes in biomarkers response associated withpollution, seasonality and latitude were evaluated.

2. Materials and methods

2.1. Sampling sites

Immature adult oysters C. rhizophorae from both sexes (4–7cm length) were sampled in winter (August/2003) and summer(February/2004) in three estuarine regions along the Braziliancoast: Paranaguá Bay (Southern Brazil), estuarine complex ofPiraquê–Mirim and Piraquê–Açú Rivers (Southeastern Brazil)and Itamaracá Bay (Northeastern Brazil) (Fig. 1). As a

consequence of the latitudinal position of the three estuarineregions, more evident seasonal changes in temperature occurs atParanaguá Bay, where August/2003 and February/2004 repre-sent winter and summer time, respectively (Lana et al., 2001). Inthe Itamaracá region, no significant seasonal changes intemperature occur, although rainy and dry seasons, endingrespectively in August and February, are observed (Medeirosand Kjerfve, 1993).

In each region, ten oysters were collected in one reference site(R), located far from potential pollution sources such asindustries, cities and agriculture, and in two polluted sites (P-Iand P-II sites) located in another water body with similarcharacteristics. P-I site is located close to pollution sources whilethe P-II site is in the same water body, but far from these sources.Altogether, three references and six polluted sites were analyzed.

In the Paranaguá region, P-I (25°19′430″S: 48°32′39″W)and P-II (25°21′050″S: 48°25′97″W) sites receive domesticdischarges of the Paranaguá City (120,000 habitants). Inaddition, P-I site is located close to both the Paranaguá harborand a phosphate fertilizer plant. The R site (25°31′271″S:48°29′690″W) is located in the Laranjeiras Bay, which isprotected natural reservation (Teodoro, 2004). In the estuarinecomplex of Piraquê–Mirim and Piraquê–Açú Rivers, P-I(19°55′00″S: 40°12′25″W) and P-II (19°56′00″S: 40°10′00″W) sites are in the Piraquê–Açú River and receive domestic andagricultural effluents while the R site (19°55′75″S: 40°12′50″W) is in the Piraquê–Mirim River, which is not affected bythese discharges (AGB, 2004). In the Itamaracá region, P-I(7°40′047″S: 34°51′486″W) and P-II (7°40′574′S: 34°50′564″W) sites are located in the Botafogo River near a paper mill andcaustic soda and chlorine factories. In addition, sugar caneagroindustrial activities are well developed around this area.The R site (7°43′042″S: 34°53′383″W) is in the Northern partof the Santa Cruz Channel, far from these sources of pollution(CPRH, 2003).

The basic water physicochemical parameters (temperature,salinity and pH) were measured at the moment of oyster'scollection in all sites. Oyster gills were dissected in situ, storedin 1.5 ml microcentrifuge tubes, and immediately maintainedon dry ice for transport to the laboratory, where they were storedat −80 °C.

2.2. Biochemical analysis

For enzymatic assays, samples were homogenized (1:4) in acold buffer solution containing 20 mMTris, 1mMEDTA, 1 mMdithiothreitol (DDT), 0.5M sucrose, 0.15 M KCl and 0.1 mMphenylmethylsulfonyl fluoride (PMSF), with the pH adjusted at7.60. Homogenates were centrifuged at 9000×g for 30 min andthe supernatant kept for biochemical measurements. Catalase(CAT) and glutathione S-transferase (GST) activities wereevaluated as previously described by Beutler (1975) and Keenet al. (1976), respectively.

For total oxyradical scavenging capacity assay (TOSC),samples were homogenized (1:4) in K-phosphate buffer(100 mM; pH adjusted to 7.4) and centrifuged at 9000×g for30 min. The TOSC assay against peroxyradicals was performed

Fig. 1. Geographical location of the reference (R) and polluted (P-I and P-II) sites at the three estuarine regions analyzed in the present study.

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on the supernatant fraction as previously described by Winstonet al. (1998). In this assay, ethylene gas formation is monitoredby gas chromatography. The reaction is performed in thepresence of the supernatant fraction, α-keto-γ-methiobutyricacid (KMBA), and 2-2′-azo-bis-(2-methyl-propamidine)-dihy-drochloride (ABAP), in K-phosphate buffer (50 mM; pHadjusted to 7.4) at 35 °C.

Enzymatic and TOSC results were normalized to totalprotein content in the supernatant fraction, which wasdetermined using a commercial reagent kit (Doles Ltda,Goiânia, GO, Brazil) based on the Biuret method.

Metallothionein-like proteins (MT) were measured aspreviously described by Viarengo et al. (1997). Measurementswere done in a microplate reader (BioTek, Vermont, USA) aftera partial purification step. Sample absorbance readings wereconverted to pmol of glutathione (GSH) using a GSH standardcurve.

Lipid peroxides level (LPO) was evaluated through the FOXassay as described by Hermes-Lima et al. (1995) and adaptedfor invertebrate tissues by Monserrat et al. (2003). Sampleswere homogenized (1:9) in 100% methanol and centrifuged at

1000×g, for 10min. The supernatant fraction was diluted (1:1.5)in 100% methanol to avoid saturation of the method. Lipidhydroperoxides were determined using FeSO4 (0.25 mM)prepared immediately prior its use, H2SO4 (0.25 mM), andxylenol orange (1 mM). Sample absorbance was read (550 nm)after 100 min of incubation at room temperature (20 °C).Sample absorbance was converted to nmol of cumenehydroperoxide (CHP) using a CHP standard curve.

2.3. Statistical analysis

All values are reported as means±standard deviation.Statistical analysis was carried out using three-way analysis ofvariance (ANOVA), considering sample site (R, P-I and P-II),season (August/2003 and February/2004) and estuarine region(Paranaguá, Piraquê and Itamaracá). Differences among siteswere checked for each estuarine region considering results forthe same season. Seasonality and latitudinal effects onbiomarkers were verified comparing only the reference sitesfor the three estuarine regions. Logarithmic transformation wasemployed if normality and/or homoscedasticity were not

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verified. A posteriori comparisons were performed with Duncantest. The significance level adopted was 95% (∝=0.05).

3. Results

3.1. Pollution-related changes in biomarkers

Oysters from the P-II at the Paranaguá Bay showedsignificantly higher CAT activity than those from the R site inwinter. However, oysters from P-I showed significantly higherCAT activity than those from the R site in summer (Fig. 2). Inthese oysters, evidence of oxidative stress was observed inwinter, since LPO level was significantly higher than that found

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Fig. 2. Catalase (CAT) and glutathione S-transferase (GST) activity, and total oxCrassostrea rhizophorae collected in winter (left panel) and summer (right panel). Esites for the same estuarine region and season (P>0.05). Equal small letters indicaestuarine regions (P>0.05). #Indicates significant seasonal difference for the same rindicates the number of oysters analyzed in each group.

in oysters from the R site. In summer, LPO level wassignificantly lower in oysters from the P-I and P-II sites thanin those from the R site (Fig. 3).

In oysters from the Piraquê estuarine region, pollution-related responses in biomarkers were only observed in summer.GSTactivity in oysters from the P-II site was significantly lowerthan that of oysters from the R site. Oysters from the P-I and P-IIsites also showed significantly lower TOSC values than thosefrom the R site (Fig. 2).

At Itamaracá Bay, GST activity was higher in oysters fromthe P-I site than in those from the R site in both seasons. Inwinter, oysters from the P-II site showed significantly lowerTOSC value than those from the R site.

erence

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yradical scavenging capacity (TOSC) values in gills of the mangrove oysterqual capital letters indicate absence of significant differences between collectionte absence of significant difference between reference sites from the differenteference site at the same estuarine region (P<0.05). The number in parenthesis

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Fig. 3. Metallothionein-like proteins (MT) and lipid peroxides (LPO) content in gills of the mangrove oyster Crassostrea rhizophorae collected in winter (left panel)and summer (right panel). Equal capital letters indicate absence of significant differences between collection sites for the same estuarine region and season (P>0.05).Equal small letters indicate absence of significant difference between reference sites from the different estuarine regions (P>0.05). #Indicates significant difference forthe same reference site at the same estuarine region (P<0.05). The number in parenthesis indicates the number of oysters analyzed in each group.

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No significant differences in the concentration of metal-lothionein-like proteins were observed between the polluted andreference sites in all estuarine regions (Fig. 3).

As showed in Table 1, water salinity, temperature and pHwere quite similar at the different sampling sites for the sameestuarine region.

3.2. Season-related changes

Seasonal changes in biomarkers were observed in oystersfrom the Piraquê and Itamaracá estuarine regions. At the

Table 1Physicochemical parameters of water at the oyster collection sites in winter(August/2003) and summer (February/2004). temp.—temperature (°C); sal.—salinity (ppt)

Season Site Paranaguá Piraquê Itamaracá

temp. sal. pH temp. sal. pH temp. sal. pH

Winter Reference 20.0 25.0 8.0 23.9 35.0 6.1 27.3 29.9 8.2Polluted I 22.0 23.0 7.9 24.1 33.5 7.1 28.0 21.7 7.8Polluted II 21.0 26.0 7.8 24.4 37.6 7.8 28.5 26.5 8.0

Summer Reference 28.0 20.0 8.2 29.7 20.8 10.1 30.4 20.7 8.3Polluted I 28.0 17.0 8.4 28.7 17.6 11.3 29.9 13.7 8.1Polluted II 30.0 20.0 7.9 28.5 23.2 13.3 30.1 21.0 8.6

Piraquê region, oysters collected in summer showed signifi-cantly higher GST activity than those collected in winter (Fig.2). However, an opposite seasonal response was observed in theLPO level, i.e., a significantly lower LPO level was observed insummer oysters than in winter ones (Fig. 3). At the Itamaracáregion, significantly higher CAT and GST activity (Fig. 2) wereregistered in oysters collected in summer than in winter.However, an opposite seasonal change was observed in TOSCvalues, i.e., significantly lower TOSC values were observed insummer than in winter oysters (Fig. 2).

Regarding the physicochemical parameters in water samplesfrom the R site, the most important seasonal changes observedwere for temperature at the Paranaguá Bay, and for salinity atthe Piraquê region (Table 1).

3.3. Latitude-related changes

For oysters from the R site, CAT activity was significantlylower in oysters from the Paranaguá region than in those fromPiraquê and Itamaracá regions in summer (Fig. 2). Asignificantly lower GST activity was observed in oysters fromthe Itamaracá region than in those from the Paranaguá region inwinter. An opposite change was observed for TOSC values,

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being significantly higher in oysters from the Itamaracá regionthan in those from the Paranaguá region (Fig. 2). In summer,higher GST activity and TOSC values were observed in oystersfrom the Piraquê region than in those from the Itamaracá region(Fig. 2).

As shown in Table 1, latitudinal differences in watertemperature were more evident in winter, specially when theR sites of the lowest and the highest latitude region werecompared, i.e., Itamaracá and Paranaguá regions, respectively.

4. Discussion

Environmental pollution monitoring studies employingbiochemical biomarkers in bivalves such as oysters and musselshave been widely reported in the literature (Nascimento et al.,1998; Bainy et al., 2000; Cajaraville et al., 2000; Cheung et al.,2001; Cheung et al., 2002; Lau and Wong, 2003; Roméo et al.,2003). However, only few studies have focused on theresponses of aquatic organisms simultaneously collected atdifferent estuarine regions along a wide latitude gradient. Infact, both seasonal- and latitudinal-related changes in biomar-kers, besides those correlated with pollution, were observed inthe present study.

4.1. Pollution-related changes in biomarkers

In the Paranaguá region, higher CATactivity was observed inoysters from the P-II site in winter and in those from the P-I sitein summer, suggesting a compensatory increase of thisantioxidant to cope with higher H2O2 generation directly orindirectly induced by contaminants present at these pollutedsites. Also, it is important to consider that higher CAT activitycan also indicate an increased peroxisomal proliferation inducedby organic xenobiotics such as PAHs (Cajaraville et al., 2000).

In winter, the higher LPO levels observed in oysters from theP-I site at the Paranaguá region points to an oxidative stresssituation in this season. Usually, increased LPO levels can belinked to decreased antioxidant levels and/or increment in ROSproduction (Almeida et al., 2004). The Paranaguá region islocated at the higher latitude among those analyzed in thepresent study, suggesting that lower temperatures in wintercould be acting synergistically with contaminants, contributingto the higher LPO levels observed in oysters from the P-I site atthis region. In agreement with this idea, other studies also haveshown lower antioxidant defenses in bivalves in winter(Viarengo et al., 1991; Power and Sheehan, 1996; Niyogi etal., 2001) and a parallel increment in LPO levels in this season(Viarengo et al., 1991; Niyogi et al., 2001; Wilhelm Filho et al.,2001). It is also known that invertebrates, such as barnacles(Tooke et al., 1985) and oysters (Pazos et al., 1996), haveincreased levels of PUFA (polyunsaturated fatty acids) in theircellular membrane in lower temperatures. Thus, it could behypothesized that this change in cellular membrane composi-tion, as a result of temperature variation, could contribute toincrease LPO levels, since unsaturated fatty acids are moreeasily prone to undergo peroxidation reactions in presence ofROS (De Zwart et al., 1998).

In summer, the higher CAT activity observed in oysters fromthe P-I site at the Paranaguá region, and an increased level ofother defenses against lipid peroxidation not measured here,could have contributed to the lower LPO levels observed inoysters from both P-I and P-II site when compared to those fromthe R site. Among other possible protective mechanisms againstlipid peroxides, Almeida et al. (2004) have recently reported theimportant role of phospholipid hydroperoxide glutathioneperoxidase (PHGPx) activity in mussels exposed to metals.Furthermore, it has also been demonstrated that the expressionof genes coding for desaturase Δ9 is down-regulated in oystersexposed to PAHs (Boutet et al., 2004). Thus, a lower content ofunsaturated fatty acids in cell membrane of oysters from the P-Iand P-II sites in summer cannot be ruled out as a possible causeof the lower levels of LPO observed in oysters from these sites.

In the Piraquê region, oysters collected in summer at the P-Iand P-II sites showed lower antioxidant competence againstperoxyradicals than those from the reference site. Other studiesalso have found lower TOSC values towards peroxyradicals inmussels from polluted sites (Regoli, 2000), as well as in musselstransplanted in polluted sites (Regoli, 2000; Camus et al., 2004).Usually, a reduced TOSC value is interpreted as a depletion oflow molecular weight scavengers consumed to neutralizeoxyradicals produced by the cells or an inhibition of enzymeactivity (Camus et al., 2004). Thus, it can hypothesized that thelower TOSC values observed in oysters from the P-I and P-IIsites could be a consequence of a decreased content of lowmolecular weight antioxidants such as GSH, uric acid andascorbic acid. It is important to note that these antioxidantsaccount for almost 70% of the TOSC value towardsperoxyradicals (Regoli and Winston, 1999; Regoli, 2000).This hypothesis is in accord with the fact that a lower GSTactivity was observed in oysters from the P-II site in summer,which could be due to a down-regulation of this enzymeassociated with a possible depletion of its substrate (GSH).

It the Itamaracá region, GST activity was higher in oystersfrom the P-I site than in those from the R site in both seasons.Other studies employing bivalves have also showed higher GSTactivity in organisms from polluted sites when compared tothose from reference sites (Burgeot et al., 1996; Bainy et al.,2000; Lau and Wong, 2003; Manduzio et al., 2004). Previousfield studies have shown a positive correlation betweencontaminant burden (PAHs, PCBs and PC) and GST activity(Cheung et al., 2001, 2002; Gowland et al., 2002). Pesticides arepossible contaminants present at polluted sites of the Itamaracáregion, since sugar cane plantations employing these com-pounds are intensively performed around this area. Thus,pesticides use with a consequent water contamination with thesecompounds could be contributing for the higher GST activityobserved in oysters from the P-I site at the Itamaracá region.Furthermore, the lower TOSC values in oysters from the P-IIsite compared to those from the R site indicate a depletion ofnon-enzymatic antioxidants by contaminant exposure in winter.

The absence of pollution-related changes in MT contentcould indicate an absence of metal contamination at the pollutedsites in all estuarine regions. However, it must be consideredthat MT polymorphism occurs in marine invertebrates and that

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the method employed here does not allow the measurement ofdifferent MT isoforms. In mussels, MT-20 isoform seems to bemore sensitive to Cd, a non-essential toxic metal, than MT-10isoform, that is believed to be involved in essential metals (Znand Cu) homeostasis and is present at higher basal levels inseveral organisms (Ivankovic et al., 2002).

4.2. Season-related changes in biomarkers

In the present study, temperature seems to be a minor factordetermining seasonal changes in biomarkers, since oysters fromthe R site at the Paranaguá region, the highest latitude regionstudied, showed no seasonal changes in biomarkers.

At the Piraquê region, oysters from the R site showed ahigher GST activity in summer than in winter. This increasedGST activity in summer was paralleled with a lowered LPOlevel. The higher GST activity could have contributed to thelower LPO level observed. This assumption is based on the factthat a higher GST activity would lead to a higher rate ofconjugation of lipid peroxidation by-products, allowing theirelimination by the cell transporter, the GS-X pump (Keepler,1999). In fact, Fukuda et al. (1997) showed that the activity ofthe GST-P isoform could be induced by the presence ofunsaturated aldehydes that are generated during lipid peroxida-tion processes, thus representing an important cellular defensemechanism against oxidative injury. Nevertheless, it is difficultto state if the differences observed between seasons in thePiraquê region are related to seasonality or due to a short-timeevent represented by the intense rainfall occurred some daysbefore oyster collection and the subsequent decrease in watersalinity and increase in water pH (Table 1). Bainy et al. (2000)also observed an increased GST activity in mussels associatedwith an elevated rainfall some days before animal collection.

In the Itamaracá region, oysters from the R site showedhigher GST and CAT activity in summer than in winter. Thisincreased enzyme activities could be a compensatory responseof the antioxidant defenses, since TOSC levels were lower inthat season. Seasonal differences observed in oysters from theItamaracá region can be related in fact to the rainfall indexes inthe different seasons, i.e., the rainy (winter) and dry (summer)seasons, when the hydrology of the estuary is strongly changed(Medeiros and Kjerfve, 1993).

As previously mentioned, seasonal changes in biomarkershave been associated with changes in physiological status ofoysters (e.g., growth and reproduction) or changes in the waterphysicochemical parameters (Sheehan and Power, 1999).However, data from the present study suggest that short-timeevents, such as an increased rainfall right before animalcollection, as well as seasonality, can be important factorsinfluencing biomarker responses in oysters.

4.3. Latitude-related changes in biomarkers

In winter, when latitudinal differences in water temperaturewere more evident, GST activity was lower in oysters from thelowest latitude region (Itamaracá region). In contrast, theseoysters showed higher TOSC values. These opposed response

of these biomarkers can be considered as a compensatorypattern in these antioxidants to maintain the redox balance ofoysters from different latitudes. In summer, when no markedlatitudinal differences in water temperature were noted (Table1), the higher GST and TOSC values observed in oysters fromthe Piraquê region could be associated with the short-time eventof intense rainfall that occurred some days before oystercollection.

Generally, the basal levels of GST activity were lower inoysters of the Itamaracá region than in those from other regions.This could be a particular characteristic of this oysterpopulation, maybe of genetic origin, which could be associatedwith the absence of significant decreases in temperatures at lowlatitudes. In contrast, increments in unsaturated fatty acidscontent in membrane of oysters from high latitudes, wheretemperature can significantly decrease in wintertime, would bea necessary physiological adaptation (Wallaert and Babin, 1993;Cuculescu et al., 1995). Parallel to this physiological response,higher levels of specific GST isoforms could be employed asimportant antioxidants, catalyzing lipid peroxide productsconjugation (Fukuda et al., 1997). From a practical point ofview, low GST activity would be useful for the detection ofenvironmental contaminants that increase the need of phase IIreactions. Future investigation on GST isoforms is needed todiscriminate what isoform is more responsive in oysters frompolluted sites of the three estuarine regions studied here,especially at the Itamaracá region.

In summary, data obtained in the present study point toimportant response of some biochemical biomarkers in oystersfrom polluted sites of the three Brazilian estuarine regionsanalyzed, indicating the need for future monitoring of thebiological effects of contaminants in the environments studied.They also point to the relevance to consider both season andlatitude as factors influencing biomarker responses in environ-mental contamination monitoring programs.

Acknowledgements

This study was developed in the scope of the “ProjetoRECOS” (Instituto do Milênio –MCT – Brazil). Authors thankall members of this project for their helpful assistance duringoyster collection and measurements of water physicochemicalparameters. We also thank Dr. Afonso C. D. Bainy (UFSC,Brazil) and Dr. Luis Felipe H. Niencheski (FURG, Brazil) fortheir helpful comments on the manuscript. Juliano Zanette is agraduate fellow from the Brazilian CNPq. José M. Monserratand Adalto Bianchini are research fellows from the BrazilianCNPq.

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