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Aquatic Toxicology 126 (2013) 414– 423

Contents lists available at SciVerse ScienceDirect

Aquatic Toxicology

j ourna l ho me p ag e: www.elsev ier .com/ l ocate /aquatox

nzymatic GST levels and overall health of mullets from contaminated Brazilianagoons

.F. Bastosa, R.A. Hauser-Davisb,∗, S.A.L. Tobara, R.C. Camposb, R.L. Ziolli c, V.L.F. Cunha Bastosa,. Cunha Bastosa

UERJ – Biology Institute, Department of Biochemistry, Av. Manoel de Abreu 444, Maracanã, 20550-170, Rio de Janeiro, RJ, BrazilPUC-Rio – Chemistry Department, Bioanalytics Laboratory, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900, Rio de Janeiro, RJ, BrazilUniversidade Federal do Estado do Rio de Janeiro – UNIRIO, Av. Pasteur, 458 – Urca, CEP: 22290-240, Rio de Janeiro, RJ, Brazil

r t i c l e i n f o

rticle history:eceived 2 March 2012eceived in revised form 24 August 2012ccepted 27 August 2012

eywords:nvironmental contaminationSTiomarkerST-mu

a b s t r a c t

Glutathione S-transferase (GST) assays in non-mammalian organisms are usually conducted inappropri-ately, since no previous standardization of the optimal concentrations of proteins and substrates andadequate pH is conducted. Standardization is a key task to adjust enzyme assays at their kinetically cor-rect maximal initial velocities, if one wants these velocities to indicate the amount of enzyme in a sample.In this paper GST assays were standardized in liver cytosol to compare seasonal GST levels in liver of mul-let from two contaminated lagoons in the Rio de Janeiro to those from a reference bay. GST potential asa biomarker of sublethal intoxication in this species was also evaluated. Mullet liver GST levels assayedwith substrates that corresponded to three different GST isoenzymes varied throughout the year. Thedifferences indicated that mullets are suffering from sublethal intoxication from contaminants in these

ST-pi lagoons. Seasonal variations of activity were relevant, since these could indicate differences in xenobi-otic input into the areas. An analysis of overall mullet health condition using a morphological index (theFulton Condition Factor) and macroscopic abnormalities corroborated the differences in GST levels, withfish from one of the sites in worse overall health condition showing lower and significantly different FCFwhen compared to the reference site. Therefore, GST standardized activity levels are useful biomarkersof environmental contamination for mullet.

. Introduction

Aquatic organisms are continuously exposed to xenobiotics andust have defensive strategies against toxic substances to survive.

atalysis by glutathione S-transferases isoenzymes (GST) is one ofhese mechanisms. GST represent a major group of detoxificationnzymes that catalyze the nucleophilic attachment of glutathioneGSH) to molecules that present an electrophilic carbon, nitrogenr sulphur atom (Hayes et al., 2005). They are a multigene fam-ly of dimeric enzymes present in all eukaryotic animal species,acteria, fungi and plants, and can be found in cytosol and in cellembrane. GST are classified based on their amino acid sequences,

inetic and immunological properties. Intracellular induction ofST activities by a variety of xenobiotics, including pesticides,olychlorinated biphenyls (PCB), metals and polycyclic aromatic

ydrocarbons (PAH) is very important to allow cells to get ridf toxicants. Many compounds that induce GST can themselvese substrates for these enzymes, or alternatively be metabolized

∗ Corresponding author.E-mail address: rachel.hauser.davis@gmail.com (R.A. Hauser-Davis).

166-445X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aquatox.2012.08.020

© 2012 Elsevier B.V. All rights reserved.

through cytochrome P-450 monooxygenation to compounds thatcan serve as GST substrates (Sheweita, 2000). Some GST classesdetoxify by conjugating GSH to specifics groups of xenobiotics,and different GST classes conjugating together contribute to form adefensive net against environmental pollutants, anti-tumor drugsand products of reactive stress (Hamilton et al., 2003; Hayes, 1994;Lien et al., 2002). The pi (�) and mu (�) classes of GST are the moststudied. Some GST isoenzymes, primarily of the pi and alpha classes,are also capable of reducing some organic peroxides, therefore alsoprotecting cells from oxidative stress (Ali et al., 2004; Leiers et al.,2003). As a result to exposition to xenobiotics, the overall GST activ-ity assayed with 1-chloro-2,4-dinitrobenzene (CDNB), which is arelatively good substrate for interacting with several GST isoen-zymes, may not change, or change very little. Significant changes inthe activities of certain GST classes or isoforms have been observed(Camacho, 2003), leading to the use of isoenzyme activity levels asgood environmental biomarkers (Otto and Moon, 1996).

Some lagoons in the urban area of Rio de Janeiro present

high concentrations of toxic chemicals and untreated sewage dis-charges, which affect the whole local trophic chain. The Rodrigode Freitas and Jacarepaguá Lagoons receive all kinds of urban andindustry discharges, as well as domestic sewage. The former has

Toxicology 126 (2013) 414– 423 415

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Table 1Calculated KM and Vmax for each substrate.

Assay Substrate Vmax (�mol min−1 mg−1) KM (mM)

Overall GST GSH 2.981 0.70Pi activity GSH 0.027 0.05Mu activity GSH 0.007 0.28Overall GST CDNB 2.870 0.36Pi activity ETHA 0.024 0.02Mu activity DCNB 0.006 1.15

F.F. Bastos et al. / Aquatic

een shown as heavily contaminated by metals and PAH (Loureirot al., 2009) while the latter, in addition to be contaminated byntermediate heavy metals and PAH, has also suffered extremeutrophication due to excessive discharges of untreated domes-ic sewage, that leads to the continual floration of toxic microalgaede Magalhaes et al., 2001).

This study conducted standardization assays in order to com-are seasonal cytosolic GST levels from livers of mullet (Mugil sp.)rom these lagoons to those of the reference site, Camorim Bay,ocated in the southwest coast of Rio de Janeiro (Seixas et al., 2012)nd evaluate GST potential as a biomarker of sublethal intoxicationn this species. Mullet were chosen because they are considered

sentinel species for metals and PAH (Andres et al., 2000; Canlind Atli, 2003; Neves et al., 2007). As several studies (Fernandest al., 2008; Laflamme et al., 2000; Norris et al., 2000; van der Oostt al., 1998) also demonstrate that fish exposed to environmen-al contamination show changes in the morphological parametershat determine and describe environmental interferences in theirrganisms, such as the Fulton condition factor (FCF) that evaluatedhe general health condition of a fish, we also used this index at theampling sites, to verify if fish condition would reflect the resultsrom the enzymatic assays.

. Materials and methods

All standards and reagents were purchased from Sigma Chem-cal Company (St. Louis, MO, USA). Standardizations assays werearried out by varying pH and protein and substrate concentrationsn order to find out the ideal standardized assay conditions.

.1. Sample processing

Thirty specimens of mullets (Mugil sp.), males and females, wereaptured per sampling campaign over two years from two urbanontaminated lagoons, Rodrigo de Freitas Lagoon and Jacarepaguáagoon, and from a reference site, the Camorim Bay at Angra doseis on the southwest coast of Rio the Janeiro.

Fish were euthanized by spine severing. Livers were immedi-tely weighted and homogenized using a Potter-Elvehjem typepparatus in four volumes of 0.1 M potassium phosphate buffer,H 7.0, containing 0.25 M of sucrose. The homogenate was cen-rifuged at 12,000 × g for 30 min and the supernatant was furtherentrifuged at 105,000 × g for 90 min. This last supernatant (theytosolic fraction) was pooled (five fish per pool) and used in theST activity assays following the method of Habig (Habig et al.,974). Protein concentrations were determined as described byeterson (1983).

.2. Standardization of the glutathione S-transferase (GST)ctivity assays

All standardization assays continuously registered conjugateormation for 3 min in a Shimadzu UV-160A spectrophotometer at5 ◦C. Reduced glutathione (GSH) was one of the substrates alwaysresent in the assay media. In addition to GSH the assay media con-ained as second substrates 1-chloro-2,4-dinitrobenzene (CDNB)or measuring the general GST activity at 340 nm, ethacrynic acidETHA) for assaying a pi-like class GST activity at 270 nm, or,2-dichloro-4-nitrobenzene (DCNB) for assaying a mu-like class

soenzyme activity at 345 nm. Product concentrations were cal-ulated using the absorptivity coefficients of 9.6 mM−1 cm−1 for

DNB; 5.0 mM−1 cm−1 for ETHA and 8.5 mM−1 cm−1 for CDNBHabig et al., 1974). All substrates were dissolved in absolutethanol, ensuring that the final ethanol concentration never sur-assed 4% in the cuvette.

Fig. 1. The effects of protein content on GST activity for overall, pi-like and mu-likeactivities isoenzymes. Data is shown as means ± SEM of a triplicate analysis of a fiveliver pooled sample.

4 Toxicology 126 (2013) 414– 423

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Fig. 2. The effects of pH on GST activity for overall, pi-like and mu-like activities

16 F.F. Bastos et al. / Aquatic

.3. Macroscopic examination and morphological indexalculation

Any macroscopically visible organ abnormalities and ectopara-ites were recorded.

The Fulton Condition Factor was calculated according toMaddock and Burton, 1998), using the following formula:

CF = 100WT

L3T

(1)

here total fish weight (WT, in grams) is compared to total fishength (LT, in cm).

.4. Statistical analyses

Statistical analyses were carried out using the Instat GraphPadoftware (San Diego, CA, USA). Statistical differences between thectivity values found in fish from the different locations in differ-nt seasons were tested using an unpaired Student’s t-test. Whenifferent values of variance were observed the Mann–Whitney testas used. The coefficients of variation never exceeded 10% for the

nzymatic assays. For the morphological index comparisons amonghe study sites data normality was tested using the Shapiro–Wilkes

. As the data showed a normal distribution, the ANOVA test wasonducted. Differences were considered significant when p < 0.05.

. Results and discussion

.1. Influence of different protein concentrations, pH andndogenous and exogenous substrates on GST activity

After conducting the standardization assays, the apparent val-es of the Michaelis constant (KM) and maximum velocity (Vmax)ere calculated (Table 1).

Optimal assay conditions were obtained for each assay (Table 2).o significant differences when using either a potassium phosphater a Tris–HCl buffer were observed. Therefore 0.1 M potassiumhosphate was chosen due to lower experimental blanks. Afterstablishing the values of Vmax and KM, GSH concentrations werehen standardized at 4 mM for the overall GST assay, correspond-ng to approximately six times the KM value, for Pi-like activitysoenzymes at 0.5 mM of GSH, approximately 10 times the KM valuend for isoenzyme mu at 2.5 mM, corresponding to approximatelyine times the KM value. Regarding the exogenous substrate, forhe overall GST assay, CDNB was standardized at 2.5 mM, corre-ponding to approximately seven times the KM value; for pi-likectivity isoenzymes at 0.2 mM ETHA, approximately 10 times theM value; and for mu-like activity isoenzymes at 5 mM DCNB, whichorresponded to only five times de KM value since this substrate isnsoluble at higher concentrations.

The effects of protein concentration (Fig. 1), pH (Fig. 2), GSHFig. 3) and exogenous substrates (Fig. 4) concentrations on GSTctivities are shown in a way that (A) shows the effect on the overallctivity; (B) shows the effect on pi-like activity and (C) the effectsn mu-like activity. Data are shown as means ± SEM of a triplicatenalysis of a five liver pooled sample.

Comparing biochemical responses between control organismsnd organisms from contaminated areas can be useful for deter-ining the quality of the ecosystem (Camargo and Martinez, 2006;

ole et al., 2004). Since fish are very diverse in both morpho-hysiological and biochemical aspects, it is necessary to conduct

iochemical studies of each species separately to allow enzymeinetics to be analyzed at the best conditions and so the enzymectivities can be properly compared. When comparing our resultsn GST activity levels with published data, we observed that assays

isoenzymes. Data is shown as means ± SEM of a triplicate analysis of a five liverpooled sample.

are usually conducted inappropriately, since no previous standard-ization of optimal concentrations of reagents, substrate and bufferhas been described. Many tests are carried out based on param-eters previously described for rats, thus not revealing the actuallevels of enzymatic activity in the fish species under consideration.For example, if we were to test the overall GST activity in mul-let cytosol and were to use the same GSH concentration used toassay activities in rat liver, 1 mM (Habig et al., 1974), we wouldbe using a overall activity concentration for mullet GST liver ofonly one and a half times its K , which would not guarantee that

M

the test was accurately measuring the velocity of the reaction. Theassay buffer of pH 7.8 used for testing the overall activities of GSTin liver of tilapia and mudfish from other studies (Gadagbui and

F.F. Bastos et al. / Aquatic Toxicology 126 (2013) 414– 423 417

Fig. 3. The effects of different GSH concentrations on GST activity for overall, pi-like and mu-like activities isoenzymes. Data is shown as means ± SEM of a triplicateanalysis of a five liver pooled sample.

Fig. 4. The effects of different exogenous substrate concentrations on GST for over-all, pi-like and mu-like activities isoenzymes. Data is shown as means ± SEM of atriplicate analysis of a five liver pooled sample.

Table 2Optimal activity conditions for each assay.

Assay Total protein (�g) Substrate Substrate concentration (mM) Buffer pH GSH concentration(mM)

Overall GST 25 CDNB 2.5 Potassium phosphate 0.1 M 7.5 4Pi activity (pi) 600 ETHA 0.2 Potassium phosphate 0.025 M 6.0 0.5Mu activity (mu) 750 DCNB 5 Potassium phosphate 0.1 M 7.0 2.5

4 Toxico

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18 F.F. Bastos et al. / Aquatic

oksoyr, 1996) would also be inappropriate, since this pH wouldause a decrease in the mullet liver activity, whose optimum pHound by us was 7.5. Varying the concentrations of endogenousnd exogenous substrates we were able to calculate the KM andmax of hepatic cytosolic fractions using CDNB, ETHA and DCNBubstrates. It is known from classical kinetics that by adjusting aubstrate concentration ten times the KM value, the enzyme wouldatalyze the reaction with a maximum speed, reflecting then themount of enzyme. Sometimes this is not feasible, because the sub-trate could present a low solubility at this concentration, as is thease for DCNB, which we used at 5 mM; only about five times higherhan the KM of 1.15 mM. The Michaelis–Menten constant (KM) is

dynamic constant that expresses the relationship between theelocity constants of all the stages of an enzymatic catalysis ands valuable in relating the speed of an enzymatic reaction with theubstrate concentration. In practice, KM is equivalent to the sub-trate concentration that gives half of the maximum velocity of

he enzyme. Thus, a lower enzyme KM indicates a higher substrateffinity of this enzyme to its substrate. Analyzing the results of theresent study, we observed that KM of the pi isoenzyme for GSH

s about six times lower (0.05 mM) than the KM for GSH of the

ig. 5. Overall, pi and mu GST activities of mullets from the Camorim Bay throughouthe year. Data is shown as means ± SEM of a triplicate analysis of at least five fish.ignificant differences (Student’s t-test and Mann–Whitney test) are indicated by *P < 0.05).

logy 126 (2013) 414– 423

isoenzyme mu (0.28 mM). Furthermore, the isoenzyme pi has aKM value 57 times smaller (0.02 mM) for its exogenous substrate,ETHA, than isoenzyme mu for DCNB (1.15 mM). This means thatisoenzyme pi needs lower substrate concentrations to reach theirmaximum efficiency for substrate metabolization. Since Vmax andKM values of an enzyme capable of catalyzing a biotransforma-tion reaction and the detoxification capacity of a tissue are related,it would be advantageous for cells to have biotransformationenzymes capable of acting with both high affinity for substratesand high effective catalytic capacity. Such enzymes could reduceorgan intoxication levels without needing effectors other than thechange in concentration of their own substrates. Thus, enzymeswith the highest Vmax and lowest KM would then have the abilityto form products more effectively. Accordingly, the efficiency withwhich a tissue conjugates GSH with a xenobiotic may depend onthose GST isoenzymes whose Vmax/KM ratio are higher. Pi-like activ-ity we have assayed here in cytosol from liver of mullet matchesthis rationale, which indicates that liver of mullets can have moreability to conjugate xenobiotics by GST pi-like isoenzymes, whatmake this activity an adequate biomarker.

3.2. Seasonal levels of GST activity in mullet livers

GST activities were compared throughout the seasons. OverallGST activity in fish from the Camorim Bay in the summer was signif-icantly high (2.0 �mol min−1 mg−1) and lower in the other seasons(Fig. 5A). In fish from the Camorim Bay pi-like and mu-like GSTactivities were significantly low only in spring (Fig. 5B and C).

In fish from the Rodrigo de Freitas Lagoon the overall GSTactivity was high throughout the year, with a significant decrease(0.7 �mol min−1 mg−1) in autumn (Fig. 6A). The pi-like activityisoenzyme of fish from the Rodrigo de Freitas Lagoon did not varythroughout the year (Fig. 6B), while the mu-like activity class insummer and, mainly, in autumn was significantly elevated whencompared to the other seasons (Fig. 6C). No significant differenceswere observed when comparing activities of fish sampled in winterand spring (Fig. 6C). GST overall activity at the Jacarepaguá Lagoonwere significant lower in autumn when compared to the othersseasons (Fig. 6D). The pi-like and mu-like activities class at this sitedid not show significant differences (Fig. 6E and F).

3.3. Comparison of GST activity levels in liver cytosol of mulletfrom the reference site with those from Rodrigo de Freitas andJacarepaguá Lagoons

When comparing the overall GST activities of mullets from thereference site to the fish of the Rodrigo de Freitas and JacarepaguáLagoons in the summer it is possible to notice that in the pollutedlagoons the mullets had less GST activity (Fig. 7A). The activitiesof pi-like GST isoenzymes were significantly decreased in both theRodrigo de Freitas and the Jacarepaguá Lagoons as compared tothe control area (Fig. 7B). There was also a reduction in the activ-ity of mu-like GST isoenzymes in fish from both study sites, butthis reduction was only significant at the Rodrigo de Freitas Lagoon(Fig. 7C).

In autumn a significant increase in the overall GST activity in fishsamples from the Rodrigo de Freitas Lagoon was observed, but notin fish from Jacarepaguá (Fig. 8A). GST pi-like activity from bothcontaminated sites are significantly lower in the autumn whencompared to the control area (Fig. 8B), and a significant increasein the mu-like activity class in fish from the Rodrigo de FreitasLagoon was also observed, while this was not observed in fish from

Jacarepaguá (Fig. 8C).

In winter, the overall GST activity increased significantly in bothcontaminated areas and the activity level of fish from Jacarepaguáis the highest among the three study areas (Fig. 9A). The increase in

F.F. Bastos et al. / Aquatic Toxicology 126 (2013) 414– 423 419

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ig. 6. Overall, pi and mu GST activities of mullets from Rodrigo de Freitas and Jacaref at least five fish. Significant differences (Student’s t-test and Mann–Whitney test

verall GST activity is not reflected in an increased activity of mu-ike and pi-like activities isoenzymes. The pi-like activity GST classn winter was significantly reduced in fish from the contaminatedreas in relation to fish from the Camorim Bay (Fig. 9B). As for mu-ike activity class, there was only a significant reduction of activityn the livers of fish from the Rodrigo de Freitas Lagoon (Fig. 9C).

In spring, the GST overall activities of fish from the Rodrigo dereitas and Jacarepaguá Lagoons are significantly higher than thosef the Camorim Bay (Fig. 10A). The increase in the overall activ-ty at the Rodrigo de Freitas Lagoon does not seem to be relatedo increased pi-like and mu-like activities, since a small activityeduction is observed in the pi-like activity GST class at the Rodrigoe Freitas Lagoon (Fig. 10B). Analyzing the results of the mu-likectivity GST class we observed a small but significant increase atacarepaguá (Fig. 10C).

Comparing the activities found in mullets collected in theutumn at Camorim Bay (Fig. 5B and C) with other Neotropi-al fish from the literature we found that the armored catfish

resents an activity (0.245 �mol min−1 mg−1) ten times greaterhan that of mullets in relation to the ETHA substrate and 49 timesigher for DCNB (0.039 �mol min−1 mg−1). On the other hand, the

Lagoons throughout the year. Data is shown as means ± SEM of a triplicate analysisndicated by * (P < 0.05).

pacu fish presents activities about six times higher for both ETHA(0.143 �mol min−1 mg−1) and DCNB (0.039 �mol min−1 mg−1)(Camacho, 2003). As pacu and catfish showed higher DCNB catalyticactivity, reflecting an elevated presence of mu isoenzymes, whichare thought to be responsible for detoxification of oxidized andepoxy forms of PAH, it is plausible that pacu and catfish are moreprepared to overcome this kind of contaminants in their habitatsthan mullets. The same could be said for xenobiotics metabolizedby pi isoenzymes by these species, since they also have high levelsof activity with ETHA.

For CDNB, a substrate that tests the activities of several GSTclasses, the KM in mullets was of 0.36 mM, while that found fortrout was of 1.8 mM (Gallagher et al., 2000). The Vmax in trout was0.014 �mol min−1 mg−1 while in mullets this value was 2.89. Whenwe calculate the efficiency of enzymatic catalysis we observe a cat-alytic efficiency (Vmax/KM) twenty times higher for mullet GST. Wepostulate that mullets, therefore, show better detoxification abili-ties when compared to trout concerning substrates of pi-like GST

isoenzymes.

Studies have also been conducted with other species in north-western Spain (Novoa-Valinas et al., 2002) that compared the total

420 F.F. Bastos et al. / Aquatic Toxicology 126 (2013) 414– 423

Fig. 7. Overall, pi and mu GST activities of mullets between the CamorimBmd

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Fig. 8. Overall, pi and mu GST activities of mullets between the CamorimBay, Rodrigo de Freitas and Jacarepaguá Lagoons in autumn. Data is shown as

ay, Rodrigo de Freitas and Jacarepaguá Lagoons in summer. Data is shown aseans ± SEM of a triplicate analysis of at least five fish. Significant differences (Stu-

ent’s t-test) are indicated by * (P < 0.05).

nd pi GST activities in Atlantic salmon and brown trout in captivity.y using 4 mM GSH (the same concentration used in our tests forverall GST) with CDNB in the cytosol of liver of Atlantic salmon theeneral GST activity (0.29 �mol min−1 mg−1) was lower than theST activities found in autumn for mullets (0.43 �mol min−1 mg−1)

Fig. 5A). Using ETHA to assay GST pi-like activity class we found velocity of approximately 0.020 �mol min−1 mg−1 along the yearFig. 5B).

Other studies which assayed the overall GST activity in eeliver using the same method that we used (Habig et al., 1974)

ithout previous standardizations of substrates concentrationsSantos et al., 2004) found approximately 20-fold lower activities0.15 �mol min−1 mg−1) than the activity observed in the presenttudy (2.87 �mol min−1 mg−1).

Some studies directly relating contaminant exposure to thectivity levels of GST have been conducted. One of these studiesEk et al., 2005) observed an activity of 0.49 �mol min−1 mg−1 withDNB in the liver cytosol of rainbow trout using 2 mM CDNB and

mM GSH at pH 7.4. These values are five times lower than thectivities found in mullets in the present study. When 100 mg kg−1

NT were injected into the coelom of these trout this activityncreased about one and a half time. It can be suggested that the

ST activity could be even greater in the injected trout, but suchn increase was not measured due to lack of substrate. Perez-opez et al. (2002) reported that trout exposed to 70 mg kg−1 ofCB showed an increase of 45% of enzyme activity with CDNB,

means ± SEM of a triplicate analysis of at least five fish. Significant differences (Stu-dent’s t-test) are indicated by * (P < 0.05).

which can be explained due to GST induction. Egaas et al. (1999)showed that the class mu liver isoenzyme in trout was the majorclass affected by PCB. An increase of 112% of general GST activity incarp liver and a decrease of 22% in gills was observed by Schmidtet al. (2005) when they were treated with PCB (10 mg L−1). Anotherstudy (Celander et al., 1993) injected �-naphthoflavone intraperi-toneally into trout and showed an increase of about two-fold ofpi GST, when compared to control trout. These findings indicatethat the increase in GST detoxifying enzyme activities is a veryeffective mechanism in different fish species. On this, some authors(Rodriguezariza et al., 1993) argue that Mugil sp. captured in coastalareas are well protected against oxidative stress, since they found asignificant increase in soluble and membrane GST in these animalsagainst high doses of organic xenobiotics.

Another study (Sole et al., 2003) compared GST activities in liverof carp collected from the Cardener river, in northeastern Spain,reported that in winter (January) liver GST activities were approx-imately two times higher than in March, May and July. Otherwise,one study also observed that female eels (Zoarces viviparus) fromthe west coast of Sweden had higher levels of overall GST activ-ity in the warmer months, August and September (Ronisz et al.,1999). We observed that the overall GST activity of mullets in

three sampling areas is higher in the summer with a decline inthe other seasons of the year in fish from Camorim Bay. However,the decrease of the overall activities of fish in the other areas were

F.F. Bastos et al. / Aquatic Toxicology 126 (2013) 414– 423 421

Fig. 9. Overall, pi and mu GST activities of mullets between the Camorim Bay,Rodrigo de Freitas and Jacarepaguá Lagoons in winter. Data is shown as means ± SEMoa

ntsettiwiowc

aCaatatiis(

Fig. 10. Overall, pi and mu GST activities of mullets between the Camorim Bay,

Freitas Lagoon, which is more heavily contaminated by metals and

f a triplicate analysis of at least five fish. Significant differences (Student’s t-test)re indicated by * (P < 0.05).

ot as pronounced in autumn, winter and spring, suggesting thathe Rodrigo de Freitas and Jacarepaguá Lagoons may be sufferingublethal effects of xenobiotics. It is important to note that the lev-ls of overall GST activity of these sites were not accompanied byhe mu and pi levels of these samples, which were low. If we hadested the activities of other isozymes, for example, class alpha,nvolved in antioxidant metabolism, they might have been higher,

hich would explain why there are high levels of the overall activ-ty. The increase in the overall activity in summer compared tother seasons was also observed in curimbatá, a freshwater species,hen they were placed in cages and subjected to streams severely

ontaminated with anthropogenic agents (Campbell et al., 2006).Fish caught in autumn showed significantly higher overall GST

ctivity in the Rodrigo de Freitas lagoon in relation to fish fromamorim Bay (Figs. 5A and 6A). This increase was accompanied byn increase in mu-like activity isoenzymes class (Figs. 5C and 6C)nd a decrease in pi-like activity class. This lagoon has been showno be impacted by higher levels of metals and PAH than thosecceptable by international standards. GST isoenzymes are ableo bind to heavy metals and thus lose their enzymatic activity. Sot is possible that the permanently low pi-like isoenzyme activity

n fish from the contaminated areas in relation to the referenceite may be due to metal contamination present in these sitesFigs 5B and 6B and E). It is also known that the Rodrigo de Freitas

Rodrigo de Freitas and Jacarepaguá Lagoons in spring. Data is shown as means ± SEMof a triplicate analysis of at least five fish. Significant differences (Student’s t-test)are indicated by * (P < 0.05).

Lagoon is one of the most polluted by metals, with higher levelsthan the Jacarepaguá Lagoon.

The GST mu class isoenzymes are involved in the biotransforma-tion of epoxides and oxides derived from the biotransformation ofbenzo[a]pyrene, a PAH. The significant increase in mu-like activityisoenzymes in fish collected in autumn was observed at the sametime as a report indicating high PAH levels in the lagoon (Loureiroet al., 2009). Thus, we assume that the liver tissue of mullets fromthis lagoon, when absorbing PAH, induced class � isoenzymes todetoxify. Taniguchi (Taniguchi, 2002) found PAH levels forty timeshigher in fish livers from Guanabara Bay when compared to thosecollected in the bay of Ilha Grande, also at Angra dos Reis, a referencearea.

3.4. Macroscopic abnormalities and morphological index analyses

Macroscopic liver abnormalities were observed, including nod-ules, tumors, a granular appearance, a yellowish tinge and visibleectoparasites. These were more frequent in fish from the Rodrigo de

organic compounds than the Jacarepaguá Lagoon. In fish from theJacarepaguá Lagoon we also observed livers with green markings,indicative of microalgae exposure. Macroscopical gill abnormalities

422 F.F. Bastos et al. / Aquatic Toxico

Table 3Weight and length of Mugil sp. specimens analyzed in the present study. Data isshown as means ± standard deviation.

Study area Weight (g) Length (cm) FCF

Reference site 307.30 ± 67.38 29.95 ± 2.21 1.18 ± 0.10

wahJrm

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Rodrigo de Freitas Lagoon 432.08 ± 245.13 35.09 ± 7.61 0.91 ± 0.14Jacarepaguá Lagoon 208.60 ± 15.07 26.05 ± 0.10 1.15 ± 0.18

ere also present, including shortened gill arches, visible ectopar-sites and frayed and pale gills. Previous analysis in our laboratoryave also shown extremely altered hepatic tissue in fish from the

acarepaguá Lagoon, presenting generalized infection with bacte-ial infestations (data not shown), another indication that the fishust already have cellular abnormalities.Weight, length and FCF of the sampled specimens are shown in

able 3. The FCF from the contaminated area was significantly lowern fish from the Rodrigo de Freitas Lagoon when compared to theontrol area. The same was also observed in fish from Jacarepaguá,lthough this difference was non-significant.

Fish body condition is routinely employed as a measure ofsh condition, since it is a simple alternative to tissue analysisSutton et al., 2000). Low or declined condition factors, are usuallynterpreted as a depletion of energy reserves, such as stored liverlycogen and body fat (Jenkins, 2004). The FCF has been exten-ively used in the literature and is a morphological indicator ofhe general health condition of fish (van der Oost et al., 1998).ssentially, this index stipulates that a value of 1 indicates excel-ent fish health, and values below 1 indicate individuals in worseondition. Therefore, fish that show a FCF close to 1 are consid-red to be in better condition than individuals with lower FCF.his index has been considered as being consistently and signifi-antly lower in contaminated areas (Eastwood and Couture, 2002;aflamme et al., 2000; Rajotte and Couture, 2002). This index haslready been used to differentiate fish from different areas regard-ng their overall health in Brazilian lagoons, which makes it ofnterest in the present study (Hauser-Davis et al., 2010, in press).he FCF in the present study allowed for distinction between con-aminated and non-contaminated mullets, since the contaminatedite of the Rodrigo de Freitas Lagoon showed significantly lowerCF than the reference area, and are, therefore, in worse condition.his index in fish from Jacarepaguá Lagoon was also lower than theeference area, and, although this difference is non-significant, ittill indicates the usefulness of using such indices for evaluatingsh health, and is especially useful in distinguishing contaminatednd non-contaminated populations. These results corroborated thenzymatic assays, which showed that fish from the contaminatedites must be suffering from sub-lethal exposure to xenobiotics.

. Conclusions

Standardizing assays is a key step and should always beonducted, in order to correctly evaluate enzyme activity anddequately compare inter-species results. Standardization shouldnclude prospective assays to adequate the chemical nature of theuffer and its pH, as well as substrate and protein (enzyme) con-entration.

The seasonal analysis of hepatic GST levels showed differencesn the overall GST levels throughout the year. Variations could belso observed in pi and mu class GST isoenzymes. Higher GST lev-ls in the summer and lower in the spring were observed for theontrol area. Both pi and mi class activities did not vary throughout

he year at the control area, except for spring, in which they wereower. Pi-like activity class in the contaminated lagoons showedower activity in autumn. In the summer no significant differencesetween the overall GST activities in the three study areas were

logy 126 (2013) 414– 423

observed, however overall GST levels in the other seasons of theyear were elevated, indicating that fish from these areas are suffer-ing from sub-lethal intoxication from the contaminants present atthe sites. The Rodrigo de Freitas Lagoon showed significantly higherpi class activity, which is in accordance with studies that indicatethe presence of high PAH and metal concentrations found in thissite. It is of interest to observe seasonal GST variations, since thesecould indicate differences in xenobiotic input in the areas. An anal-ysis of the Fulton Condition Factor and macroscopic abnormalitiescorroborated the differences in enzymatic levels, with fish fromthe Rodrigo de Freitas Lagoon in worse overall health condition,showing lower and significantly different FCF when compared tothe reference site.

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