Ecotoxicology and Environmental Safety 57 (2004) 290–302

ARTICLE IN PRESS

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0147-6513/0

doi:10.1016

Enzymatic and nonenzymatic antioxidants as an adaptationto phagocyte-induced damage in Anguilla anguilla L. following

in situ harbor water exposure

Iqbal Ahmad, M. Pacheco, and M.A. Santos�

Animal Physiology/Ecotoxicology Sector, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

Received 1 August 2002; received in revised form 18 March 2003; accepted 28 April 2003

Abstract

Anguilla anguilla L. were caged for 8 and 48 h in harbor water of Aveiro Lagoon, Portugal. Respiratory burst activity (RBA) of

peritoneal, head kidney, and gill phagocytes was measured. Lipid peroxidation (LPO) was estimated in gill, kidney, and liver. Liver

ethoxyresorufin-O-deethylase (EROD) activity, cytochrome P450 (Cyt P450) content, and bile metabolites were assayed. Various

antioxidant enzymes, viz., glutathione peroxidase, catalase, and glutathione S-transferase and nonenzymatic antioxidant, viz., total

reduced glutathione were also studied. Harbor water xenobiotics induced a significant RBA increase in gill after 8 h; whereas in

peritoneum and head kidney it increased after 48 h exposure. These responses were adversely associated with tissue-specific

peroxidative damage since significant LPO increase was observed in gill (8 and 48 h), kidney (48 h), and liver (48 h). The tissue most

affected was gill. Moreover, liver EROD activity, Cyt P450 content and bile metabolites remain unaltered after 8 h; in contrast, 48 h

exposure showed significant EROD activity decrease and pyrene-type bile metabolites increase. Decreased EROD activity may be

associated with concomitant liver damage, as increased LPO was observed after 48 h. Furthermore, the tissue-specific damage

corresponded to the differences in the antioxidant potentials of the tissues, since the initial exposure period caused a significant

increase in liver antioxidant activities, whereas gill and kidney showed a significant decrease, demonstrating that liver is highly

adaptive to oxidative damage. However, at 48 h exposure gill, kidney, and liver showed a suppressive antioxidant effect, probably

due to PAHs, since a significant induction at PAH-type bile metabolites has been seen. Our results demonstrate that phagocyte

activation and associated peroxidative damage are concomitantly corroborated with enzymatic and nonenzymatic antioxidant

activity differences. In addition, hepatic antioxidant induction after short-term exposure may serve as a potent biomarker for water

pollutants in fish.

r 2003 Elsevier Inc. All rights reserved.

Keywords: Anguilla anguilla L.; Phagocytes; Respiratory burst activity; Harbor water contamination; Peroxidative damage; Antioxidants

1. Introduction

An increasing variety of industrial, agricultural andother chemicals are entering the aquatic environmentand being taken up into the tissues of invertebrates andvertebrates (Ahmad et al., 2000; Khan et al., 1995;Walker and Livingstone, 1992; Walsh and O’Halloran,1988). The uptake of these contaminants can occur fromsediments, suspended particulate materials, water col-umn and food sources. The major route of inputdepends on the particular dietary and ecological lifestyles of the organisms (Livingstone, 1991, 1998; Van

onding author. Fax: +351-234426408.

ddress: monteiro@bio.ua.pt (M.A. Santos).

3/$ - see front matter r 2003 Elsevier Inc. All rights reserved.

/S0147-6513(03)00080-0

Veld, 1990). Such a diverse array of chemicals can havemany different mechanisms of toxicity and severalmechanisms may exist for a single contaminant, eachcontributing to various degrees of the final overalldeleterious effects (Livingstone, 1991; Livingstone et al.,2000; Walker et al., 1996). The cytotoxic reactive oxygenspecies (ROS) are continuously produced by contami-nant-stimulated phagocytes (Ahmad et al., 1998; Fatimaet al., 2000) and excessive ROS have shown their role inthe development of local tissue damage (Ahmad et al.,2003; Fatima et al., 2000). However, there is a lack ofinformation on the defense mechanisms that neutralizethe impact of ROS on fish. The exclusive emphasis ofprevious research work has been centered on ROSdeleterious capacity for biological macromolecules

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(Girotti, 1990; Stadtman, 1991) and local tissues, wherethey home in (Ahmad et al., 2003; Fatima et al., 2000).The importance of radical reactions in normal biologicalprocesses and in the mechanisms of toxicity of manyforeign compounds has given rise to a research thrustinto cell pro-oxidant and antioxidant processes (Halli-well and Gutteridge, 1999; Kehrer, 1993). The level ofantioxidants against ROS overproduction and itsassociation with local tissue damage can be interpretedas a toxicity measure in aquatic organisms exposed topollution (Ahmad et al., 2000; Di Giulio et al., 1989,1995; Filho, 1996; Lackner, 1998; Lemaire and Living-stone, 1993; Lopez-Torres et al., 1993; Winston andDi Giulio, 1991).Previously, it has been shown that overactivation of

fish phagocytes was associated with the release of ROS,contributing to the overall oxidative stress in fish tissuesinflicted by the constituents of paper mill effluent and bysublethal naphthalene exposure in laboratory conditions(Ahmad et al., 1998, 2003; Fatima et al., 2000).Additionally, we have also observed that this differentiallipid peroxidation (LPO) response was due to differentantioxidant levels in tissues of exposed animals (Ahmadet al., 2000). As several laboratory studies have beendone in order to observe the role of circulating fishphagocytes, we were interested to observe this approachunder natural environmental conditions on the AveiroLagoon harbor water. Our main aim in the presentstudy was to understand the relationship betweendifferent biological responses, viz., overactivated pha-gocytes vs peroxidative damage, its association to theantioxidant enzymes and nonenzymatic antioxidantdefense molecules and liver biotransformation capacityon natural Aveiro Lagoon harbor water. In this work wehave studied phagocytic respiratory burst activity(RBA) for ROS production; tissue oxidative stressestimators as gill, kidney, and liver LPO; liver biotrans-formation indicators such as ethoxyresorufin O-deethy-

Fig. 1. Representation of A. anguilla sampling area and exposure cag

lase (EROD) activity and cytochrome P450 (Cyt P450)content; bile metabolites; antioxidant enzymes: glu-tathione peroxidase (GPX), catalase (CAT), and glu-tathione S-transferase (GST), and nonenzymaticantioxidant such as glutathione (GSH) in gill, kidney,and liver of fish.

2. Materials and methods

2.1. Chemicals

1-chloro-2,4-dinitrobenzene (CDNB), dimethyl sulf-oxide (DMSO), 2,5-dithio-bis-tetranitrobenzoic acid(DTNB), Hank’s balanced salt solution (HBSS), histo-paque-1077, nitroblue tetrazolium (NBT), Roswell ParkMemorial Institute-1640 (RPMI-1640), sigmacote, 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA),resorufin, and dithionite were purchased from SigmaUSA, NADPH and 7-ethoxyresorufin were from Roche,and other routine chemicals and reagents (analyticalgrade) were purchased from local sources.

2.2. Animals

European eel, Anguilla anguilla L., of length26.1273.2 cm and weight 33.6711.3 g, were capturedat the Aveiro Lagoon (Fig. 1) and were acclimatized for7 days under standard conditions (APHA, 1998) in 80-Lartificial sea water aquaria (Sera Meersaltz, Heinsberg,Germany). Water oxygen saturation was maintainedby aeration. Water temperature was maintained at2071.4�C.

2.3. Environmental contaminated water exposure

A. anguilla were caged into a harbor area of the AveiroLagoon (Fig. 1) for 8 and 48h, after acclimatization,

ing site (harbor water) on the Aveiro Lagoon, Aveiro, Portugal.

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without any food supply. During acclimatization thewater salinity was maintained according to the harborwater salinity. The caging site is located in an offwardfishing harbor. Two groups of 10 fish each weretranslocated from the laboratory to the exposure sitein a recipient without water (30min) placed, in 80 dm3

net cages, and plunged in the harbor water near thesediments. For control, two more groups of 10 fisheach were maintained in the laboratory at 20�C in asimilar net cage and submitted to the equivalentprevious dry period (30min). The experiment wascarried out in September, at approximately 20�C watertemperature.

2.4. Experiment

The objective of this study was to evaluate the roleof various antioxidant enzymes and nonenzymaticantioxidants against circulating fish phagocytes inducedtoxicity in the natural conditions of Aveiro Lagoonharbor water. These objectives were based on theinformation reported in our previous laboratory studyin A. anguilla concerning naphthalene exposure (Ahmadet al., 2003). A. anguilla showed measurable significantaffects at 8 and 48 h exposure to naphthalene (Ahmadet al., 2003). Keeping in view these preliminaryobservations the field study was conducted by plungingcaged A. anguilla into harbor water for 8 and 48 h.

2.4.1. Isolation of phagocytes from peritoneum cavity,

gill and head kidney

Phagocytes were isolated using our previously de-scribed method (Ahmad et al., 1998). Briefly, the fishblood was drained by severing the caudal vein to avoidthe contamination of peritoneal and gill exudates by redblood cells. An abdominal incision was made and theperitoneal cavity and gill were thoroughly rinsed withcold HBSS in siliconized test tubes, transferred intoseparate unsiliconized Petri dishes, and incubated for 1 hfor adherence. These dishes’ supernatant was decantedand cells were detached with the help of a cell scraper infresh cold HBSS. Detached cells were centrifuged insiliconized tubes at 2500 r.p.m. for 10min (� 3) and thefinal cell pellet was resuspended in 1mL of RPMI-1640medium. Cells were counted using a hemocytometer.Differential counts were performed to assess thephagocyte populations in the cell suspensions. Fishhead kidney (pronephros), a hemopoietic and lymphoidtissue, was dissected out of the fish and minced using acell dissociation sieve tissue grinder kit (Sigma). Thecells were separated from tissue in RPMI-1640. Thesuspension was layered onto Histopaque-1077 andcentrifuged at 1800 r.p.m. for 30min. The upper celllayer was collected, washed twice with RPMI-1640, andused for experiments.

2.4.2. RBA assay

The oxidative burst phenomenon was studied toobserve the activation pattern of phagocytes in differentfish groups. RBA of peritoneal cavity, gill and headkidney fish phagocytes was measured by the method ofFujiki and Yano (1997), modified by Fatima et al.(2000). Briefly, an aliquot (0.1mL) of phagocytesuspension was mixed with 0.5mL HBSS-P containing0.1% NBT and 25� 106 cells/mL yeast cells (heattreated at 100�C for 1 h). The mixture was incubatedat 35�C for 2 h with occasional shaking. The suspensionwas centrifuged at 3000 r.p.m. for 15min after incuba-tion. The cell pellet was washed with 70% methanol,air-dried at 60�C for 15min, and solubilized with 0.5mLof 2M KOH by sonication in an ultrasonic processor(U 200 S control, Germany) for 5min. The sonicatedmaterial was extracted with 0.5mL DMSO for 5min.The NBT reduction potential of phagocytes wasmeasured in the supernatant after centrifugation at4500 r.p.m. for 5min as absorbance at 630 nm andexpressed as A630 nm per 106 cells.

2.4.3. Estimation of LPO in fish tissues

LPO was estimated in gill, kidney, and liver of treatedand control fish. Gill were excise out of the sacrificedfish, gill rackers were removed, and gill lamellae werehomogenized in a Potter–Elvehjem homogenizer. TheLPO activity was determined by the procedure of Utleyet al. (1967) modified and adapted by Fatima et al.(2000). Briefly, the tissues were homogenized in chilled0.15M potassium chloride solution. Thereafter 1.0mLof each homogenate was dispensed in a glass tube andincubated at 37�C in a metabolic shaker for 60min. A1.0mL aliquot of the same tissue homogenate wasincubated at 0�C, which served as a control reaction set.After 1 h incubation, 1. 0mL of 10% chilled TCA and1.0mL of 0.67% TBA were added to each sample andmixed well. The aliquots of each vial were thencentrifuged at 3000 r.p.m. for 10min. The supernatantfrom each tube was decanted into separate tubes andplaced in a boiling water bath. The test tubes wereremoved and cooled at room temperature after 10minboiling. The absorbance of each aliquot was measuredat 535 nm. The rate of LPO was expressed as nanomolesof thiobarbituric acid reactive substance (TBARS)formed per hour per milligram of protein using a molarextinction coefficient of 1.56� 105M�1 cm�1.

2.4.4. Liver EROD assay

Microsomes were obtained according to the methodsof Lange et al. (1993) and Monod and Vindimian(1991), adapted by Pacheco and Santos (1998). ERODactivity was measured as described by Burke and Mayer(1974). The reaction was carried out in a fluorometercuvette containing 1mL 0.5 mM ethoxyresorufin (0.1MTris-HCl buffer, pH 7.4, containing 0.15M KCl and

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20% glycerol) and 25 mL of microsomal suspension at25�C with constant agitation and initiated by adding10 mL of NADPH (10mM). The progressive increase influorescence resulting from the resorufin formation wasmeasured for 3min (excitation wavelength 530 nm,emission wavelength 585 nm) with a Jasco FP-777spectrofluorometer. Results were expressed as pmol/min/mg of microsomal protein.

2.4.5. Cyt P450 assay

Liver microsomal Cyt P450 content was determinedusing the dithionite reduced carbon monoxide differencespectrum between 450 and 490 nm, as previouslydescribed by Hermens et al. (1990).

2.4.6. Fixed wavelength fluorescence (FF) of bile

FF of bile was performed according to the methods ofAas et al. (1998), Lin et al. (1996), and Gagnon andHoldway (2000). FF readings discriminate between2-, 4-, and 5-ring PAH metabolites, i.e., naphthalene-,pyrene-, and benzo(a)pyrene-type metabolites by usingthe wavelengths pairs 290/335, 341/383, and 380/430 nm, respectively. Bile samples were diluted with 20volumes of water to give an initial bile solution that wasassayed for protein content. This solution was furtherdiluted in methanol 50% to 1:400 for FF measurement.A 5-nm slit width was used for both excitation andemission. Bile metabolites are reported on the basis ofmg protein.

2.4.7. Preparation of post-mitochondrial supernantant

(PMS)

PMS was obtained according to Ahmad et al. (2000).Liver, kidney, and gill of the sacrificed fish were quicklyremoved, cleaned of extraneous material, and immedi-ately perfused with ice-cold saline. The tissues werehomogenized in chilled phosphate buffer (0.1M, pH 7.4)containing KCl (1.17%), using a Potter–Elvehjemhomogenizer. The homogenate was filtered throughmuslin cloth and centrifuged at 800 g for 5min at 4�Cto separate the nuclear debris. The supernatantwas centrifuged in a refrigerated ultracentrifuge (OptimaTL, Beckman) at 10,000 r.p.m. for 30min at 4�C toobtain the PMS, which was used for biochemicalanalysis.

2.4.8. GPX activity

GPX activity was assayed according to the methoddescribed by Mohandas et al. (1984) as modified byAthar and Iqbal (1998). The assay mixture consisted of1.44mL phosphate buffer (0.05M, pH 7.0), 0.1mLEDTA (1mM), 0.1mL sodium azide (1mM), 0.05mLGSH reductase (1 IU/mL), 0.1mL GSH (1mM),0.1mL NADPH (0.2mM), 0.01mL H2O2 (0.025mM),and 0.1mL of PMS (10%) in a total volume of 2mL.Oxidation of NADPH was recorded spectrophotome-

trically (Jasco UV/VIS, V-530) at 340 nm at roomtemperature. The enzyme activity was calculated asnanomoles NADPH oxidized/min/mg of protein, usinga molar extinction coefficient of 6.22� 103M�1 cm�1.

2.4.9. CAT activity

CAT activity was assayed by the method of Claiborne(1985) as described by Giri et al. (1996). Briefly, theassay mixture consisted of 1.95mL phosphate buffer(0.05M, pH 7.0), 1mL hydrogen peroxide (0.019M),and 0.05mL 10% PMS in a final volume of 3mL.Change in absorbance was recorded at 240 nm. CATactivity was calculated in terms of nanomoles H2O2

consumed/min/mg protein.

2.4.10. GST activity

GST activity was determined by the method of Habiget al. (1974) with some modification (Raisuddin et al.,1994). The reaction mixture consisted of 1.65mLsodium phosphate buffer (0.1M, pH 6.5), 0.1mLreduced GSH (1mM), 0.05mL CDNB (1mM), and0.2mL PMS (10%) in a total volume of 2mL. Thechange in absorbance was recorded at 340 nm andenzyme activity calculated as nanomoles CDNB con-jugate formed/min/mg protein using a molar extinctioncoefficient of 9.6� 103M�1 cm�1.

2.4.11. Estimation of GSH

GSH was determined in PMS by the method ofJollow et al. (1974) as described by Iqbal et al. (1999).PMS (1mL) was precipitated with 1mL of sulfosalicylicacid (4.0%). The samples were kept at 4�C for 1 h andthen subjected to centrifugation at 3000 r.p.m. for15min at 4�C. The assay mixture contained 0.5mL offiltered aliquot, 2.3mL of sodium phosphate buffer(0.1M, pH 7.4), and 0.2mL DTNB (stock=100mM in0.1M sodium phosphate buffer, pH 7.4) in a totalvolume of 3mL. The reaction product optical density(OD) was read immediately at 412 nm on a spectro-photometer.

2.4.12. Protein measurement

Total protein content was determined according tothe Biuret method (Gornall et al., 1949), using bovineserum albumin (E. Merck-Darmstadt, Germany) as astandard.

2.5. Statistical analysis

The means significance difference between controland exposed animals was determined using the two-tailed Student’s t-test. The significance level was set atPo0.05.

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Fig. 2. RBA in peritoneum, gill and head kidney phagocytes of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values

are expressed as OD at 630 nm/106 cells (mean7SE, n=5/exposure). The significance levels observed are �Po0.05 and ��Po0.01 when compared

with their respective control values.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302294

3. Results

3.1. Effect on RBA of fish phagocytes

Fig. 2 shows the RBA as measured by the NBTreduction of fish phagocytes isolated from peritoneum,gill, and head kidney after 8 and 48 h exposure tocontaminated harbor water. No significant RBA induc-tion was observed after 8 h exposure, whereas 48 hexposure showed significant (Po0.05) RBA induction inperitoneal exudate phagocytes. In gill-adhered phago-cytes, the induction was significantly (Po0.01) pro-nounced at 8 h exposure. A similar significant (Po0.01)RBA induction was observed in head kidney phagocytesafter 48 h exposure; in contrast, 8 h exposure showed nosignificant change in RBA when compared with therespective controls.

3.2. Effect on LPO in fish tissues

Contaminated harbor water exposure increased LPOactivity significantly (Po0.001, Po0.05) in gill after 8and 48 h exposure (respectively) (Fig. 3). A significantly(Po0.01) increased LPO was also found in kidney andliver after 48 h exposure to harbor water. However, 8 hexposure showed no significant change in LPO activityin either kidney or liver.

3.3. Effect on liver EROD activity

Results in Fig. 4 reveal that harbor water is able toinduce a significant (Po0.05) EROD activity decrease inliver after 48 h exposure and a nonsignificant decreasein its activity after 8 h exposure.

3.4. Effect on liver Cyt P450 content

This biomarker exhibited no statistically significantalteration in liver after 8 or 48 h exposure to harborwater (Fig. 5), despite the slight decrease observed inexposed fish.

3.5. Effect on fixed wavelength fluorescence (FF) of bile

Exposed fish exhibit a significant (Po0.05) increase inpyrene-type bile metabolites after 48 h exposure (Fig. 6).Additionally, benzo(a)pyrene-type metabolites increasedconsiderably after 8 and 48 h exposure, despite theabsence of any statistical significance.

3.6. Effect on enzymatic antioxidants in fish tissues

Harbor water exposure showed a significant (Po0.01)decrease in gill GPX activity after 8 and 48 h of exposure(Fig. 7). The decrease was 44.19% and 51.55%,respectively. A significant (Po0.01) decrease in GPX

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Fig. 3. LPO values in gill, kidney and liver of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values are expressed

as nanomoles of TBARS released during 1 h of incubation (mean7SE, n=5/exposure). The significance levels observed are �Po0.05, ��Po0.01,

and ���Po0.001 when compared with their respective control values.

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Fig. 4. Liver EROD activity of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values are expressed as mean7SE

(n=5/exposure). The significance level observed is �Po0.05 when compared with control value.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302 295

activity (40.58%) was recorded in kidney after 48 hexposure (Fig. 7). Liver showed a significant (Po0.01)increase (150.10%) in GPX activity after 8 h exposure,in contrast a significant (Po0.01) decrease (32.1%) in itsactivity was observed at 48 h exposure when comparedwith control groups.Gill and kidney show a significant (Po0.05 and

Po0.01) decrease in CAT activity after 8 and 48 hexposure (Fig. 8). The decrease in gill was 35.45% and

31.22%; in kidney it was 46.73% and 45.33%. However,CAT activity was significantly (Po0.01) increased inliver (58%) after 8 h exposure.GST activity was significantly (Po0.05, Po0.01)

decreased in gill and kidney after 8 and 48 h exposure,whereas liver showed a significant (Po0.05) increase inits activity only after 8 h exposure. After 48 h exposureno significant change in liver GST activity was observed(Fig. 9). The decrease in GST activity was 47.5% and

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Fig. 5. Liver Cyt P450 content of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values are expressed as mean7SE (n=5/

exposure). The data were statistically insignificant when compared with control value.

Fig. 6. Bile PAH-type metabolites of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values are expressed as mean7SE

(n=5/exposure). The significance level observed is �Po0.05 when compared with control value.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302296

34.42% in gill; 38.07% and 36.88% in kidney; and inliver GST activity increase was 25.01%.

3.7. Effect on nonenzymatic antioxidants in fish tissues

Fig. 10 shows the total GSH contents in various A.

anguilla tissues. Harbor water significantly increasedkidney and liver GSH content, respectively, to 30.47%(Po0.05) and 34.15% (Po0.01) over control after 8 hexposure, whereas 48 h exposure induced a significant(Po0.05–Po0.001) decrease in gill (80%) and kidney(75.26%) GSH content. However, no significant effectwas observed in gill after 8 h and liver after 48 hexposure to harbor water.

4. Discussion

The present study confirms and strengths the previousstudy concerning A. anguilla phagocytes response such

as RBA to contaminants interacting with the cellmembrane. O2

�, H2O2, anddOH radicals were found

to be produced by either resident or elicited peritonealexudates phagocytes as measured by RBA and exhibitedoxidative responses in A. anguilla after exposure to theAveiro Lagoon harbor water. Historically, the harborarea has been the main receptor of anthropogenicdischarges resulting from harbor and dry dock activitiesas well as domestic effluents. This area has already beencharacterized as a complex mixture of inorganicpollutants such as heavy metals, polycyclic aromatichydrocarbons (PAHs), and organotin compounds(Abreu et al., 2000; Barroso et al., 2000; Lucas et al.,1986; Pacheco and Santos, 2001). In this study, thewater pollutants not only activated the fish phagocytesbut also developed different antioxidant potential statusagainst phagocyte-induced peroxidative damage in gill,kidney and liver. The release of large amounts of ROSby activated phagocytes and its association withperoxidative damage may become a potential cause of

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Fig. 7. GPX activity in PMS of gill, kidney, and liver of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values

are expressed as mean7SE (n=5/exposure). The significance level observed is ��Po0.01 when compared with control value.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302 297

these differences in antioxidants besides the directoxidative damage inflicted by the constituents of harborwater. Fish oxidative stress, as well as its antioxidantpotential, differs in relation to species, habitat, andfeeding behavior. For example, sole from a pollutedharbor showed a high LPO (Di Giulio et al., 1993) andelasmobranches showed higher LPO than marine tele-osts, which had much higher values than freshwaterteleosts (Filho, 1996). LPO tends to be lower inherbivorous fish than in omnivorous fish species, whichcould be correlated to the lower GPX and CATactivities (Radi et al., 1985). On the other hand,herbivorous fish species have higher superoxide dis-mutase activities than to omnivorous fish (Radi et al.,1985). When compared with other vertebrates, fishshowed similar peroxidation levels (Lopez-Torres et al.,1993). Tissue differences in the oxidative stress responsesand antioxidants are also reported in various fish species(Di Giulio et al., 1989; Mather-Mihaich and Di Giulio,1991; Radi and Matkovics, 1988).In our research work we found that harbor water

exposure has a deleterious membrane-damaging effecton gill, kidney, and liver since after an initial exposureperiod (8 h), the LPO response was significantlyincreased in gill, but not in kidney and liver. However,

48 h exposure induced a significant LPO increase ingill, kidney, and liver, which may be due to directperoxidative action by the harbor water xenobiotics.However, oxidative stress induced by ROS released bythe overactivated phagocytes also seems to contribute tothis time- and tissue-specific damage since significantRBA induction in gill and head kidney phagocytes hasbeen observed in this study. We also observed that theperoxidative damage relative to the oxidative burst wasabnormally high in these organs, perhaps due to asignificant population of circulating fish phagocytes(Ahmad et al., 1998, 2003). Laboratory experimentsconcerning Heteropneustes fossilis and A. anguilla

exposure, respectively, to paper mill effluent andnaphthalene (Ahmad et al., 2003; Fatima et al., 2000)showed similar responses in gill and kidney. RBAinduction and its association with LPO, as observed ingill and kidney may be attributed to the type, entry, andinteraction of pollutants present in the Aveiro Lagoonharbor water. Moreover, the induction of RBA andassociated peroxidative damage in gill from shortexposure (8 h) seems to result from its contiguousexposure to harbor water full of antigenic and particu-late materials. Earlier studies have confirmed that theprimary route of hydrocarbon uptake from the water

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Fig. 8. Catalase activity in PMS of gill, kidney, and liver of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values

are expressed as mean7SE (n=5/exposure). The significance levels observed are �Po0.05, ��Po0.01, and ���Po0.001 when compared with

their respective control values.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302298

occur via the gill, a very dynamic organ directly exposedto contaminated waters (Thomas and Rice, 1981).Biotransformation reactions viz EROD activity

and Cyt P450 content were focused on liver only,since gill and kidney showed minor activity changesin a previous study (Pacheco and Santos, 2001). Atthe initial exposure period (8 h) an unaltered liverEROD activity and a significant GST activity increasewere observed, revealing that the liver detoxificationprocesses were not initially depressed. After a longexposure (48 h) a clear EROD activity inhibition andunaltered GST activity in liver were observed, demon-strating a metabolic impairment, probably as a con-sequence of tissue damage. However, no statisticallysignificant change was observed in liver Cyt P450content during the whole exposure period. The GSTactivity decrease in gill and kidney after 8 and 48 hexposure, as observed in this study, indicates thevulnerability of these organs toward the harbor waterpollutant-induced toxicities. These 48 h findings couldbe due to the accumulation of PAHs in A. anguilla

tissues, since an appreciable amount of PAH-type bilemetabolites has been observed in this study. Themetabolites are excreted into the gallbladder bile and

stored there until the bile is emptied into the intestinaltract, usually during food digestion (Varanasi, 1989).Since fish were not fed during the current in situexposure, bile release is less likely. Therefore, theincreased levels of PAH-type metabolites found in bileafter 48 h exposure should be regarded as a consequenceof effective detoxification carried out by the hepatocytesin the initial exposure hours.The differences in oxidative-stress-induced antioxi-

dant potential of these organs may be the cause of theabove different responses. As in mammals, the liver isthe major site of metabolism and deposition ofxenobiotics in fish (Walker, 1980). Induction of hepaticmixed function oxidase enzymes of phase I anddetoxification enzymes of phase II biotransformationis considered a common indicator in aquatic organismsof exposure to pollutants such as PAHs. Activation ofthe antioxidant system in response to pollutant exposureis also reported in various fish species (Ahmad et al.,2000; Filho, 1996; Hai et al., 1997). Similarly, theinductive effect of paper mill effluent exposure on theantioxidant system of the freshwater fish Channa

punctatus was recently demonstrated (Ahmad et al.,2000). Currently, we observed the sensitivity of

ARTICLE IN PRESS

0

50

100

150

200

nm

ol C

DN

B f

orm

ed/m

in/m

g p

rote

in

gill

* **

0

100

200

300

400

nm

ol C

DN

B f

orm

ed/m

in/m

g p

rote

inkidney

** **

0

125

250

375

500

nm

ol C

DN

B f

orm

ed/m

in/m

g p

rote

in

8 hrs 48 hrs

Control Exposed

liver *

Fig. 9. GST activity in PMS of gill, kidney, and liver of A. anguilla L. exposed to Aveiro Lagoon harbor water for 8 and 48 h. The values

are expressed as mean7SE (n=5/exposure). The significance levels observed are �Po0.05 and ��Po0.01 when compared with their respective

control values.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302 299

enzymatic and nonenzymatic biomolecules to harborwater exposure.GPX activity decreased in gill after 8 h exposure,

corroborating the LPO increase in this organ. Thisdemonstrates the inefficiency of this organ in neutraliz-ing the impact of peroxides, resulting in increased LPO.However, for the same exposure GPX activity wasfound to be increased in liver. According to Aksnes andNjaa (1981), fish, being more susceptible to oxidativedamage, have high GPX activity in general, whereas inrainbow trout GSH-depleted liver, no peroxide-stimu-lated GSH disulfide release has been observed (Bell et al.,1986). The present study concerning 8 h exposure toharbor water shows that A. anguilla liver GPX activityincrease is possibly due to increased H2O2 production aswell as enzyme-inducing effects, whereas 48 h exposureshows a suppression of GPX activity, which may beattributed to the long-term influence of various organicand inorganic redox-active components in the harborwater.Our results demonstrate that the harbor water

exposure decreases the CAT activity in gill and kidneyand increases its activity in liver after 8 and 48 hexposure. The decrease in CAT activity observed in gill

and kidney could be due to the superoxide radicals,which have been reported to inhibit catalase activity(Kono and Fridovich, 1982). As regards liver, metal-lothionein (MT) formation is prevalent in fish (Roesjadi,1993; Tort et al., 1996). MT is also reported to scavengereactive oxygen intermediates. Although MT formationin fish gill and kidney is reported by some workers, liveris more actively involved in the formation and storage ofMT compared with gill and kidney (Hogstrand andHaux, 1996; Hogstrand et al., 1996; Marr et al., 1996;Roesjadi, 1993). In our previous investigation, protec-tion from LPO due to Cu-induced MT in the liver wasobserved, whereas kidney and gill showed no significantMT induction (Ahmad et al., 2000). This might haveincreased the vulnerability of gill and kidney toperoxidative damage by oxygen radicals on one handand the liver resistance on the other. In the presentstudy, significant liver catalase activity induction after8 h harbor water exposure could be associated with theformation of MT.GSH composes the bulk of nonprotein thiol content

of mammalian cells and has a central role in maintainingtheir normal metabolic transport function (Kosowerand Kosower, 1978). It is also important for the

ARTICLE IN PRESS

0

0.01

0.02

0.03

0.04

nm

ol/g

tis

sue

gill

*

0

0.025

0.05

0.075

nm

ol/g

tis

sue

kidney

***

*

0

0.05

0.1

0.15

nm

ol/g

tis

sue

8 hrs 48 hrs

Control Exposed

liver**

Fig. 10. Effect of Aveiro Lagoon harbor water on reduced glutathione content in gill, kidney, and liver of A. anguilla L. exposed to Aveiro Lagoon

harbor water for 8 and 48 h. The values are expressed as mean7SE (n=5/exposure). The significance levels observed are �Po0.05, ��Po0.01,

and ���Po0.001 when compared with their respective control values.

I. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302300

detoxification and excretion of xenobiotics and forprotecting cells against their toxic actions (Otto andMoon, 1995; Regoli and Principato, 1995). We foundan initial increase in A. anguilla liver and kidney GSHlevels exposed to harbor water. However, the liverlevel decreased to the control level after 48 h exposure,whereas it significantly decreased in kidney and gill. Thisbiphasic response is considered an adaptation toa detoxification activity increase (Buchner et al., 1993;Chatterjee and Bhattacharya, 1984). Moreover, theprotective effect of GSH was attributed to its ability tostablize the pollutants by scavenging the free radicalsand thereby blocking LPO development (Meister andAnderson, 1983). The initial elevated level of GSHappears to be effective in providing protection toorganisms since no significant increase in LPO wasobserved in the liver and kidney after 8 h exposure.Increased liver and kidney GSH level serves as anindicator of degree and duration of fish exposure tooxidant pollutants. The protective and adaptive role ofGSH against oxidative stress-induced toxicity is wellestablished in aquatic animals (Otto and Moon, 1995;Regoli and Principato, 1995).

This investigation establishes that pollution-inducedphagocyte overactivation may not only affects thenormal immune function but also affect other organsstructurally and functionally. The release of ROS byoveractivation of fish phagocytes after exposure toharbor water corroborates the tissue-specific peroxida-tive damage in gill, kidney, and liver after 8 h. The tissuemost affected was gill. This finding also establishesthe development of tissue-specific adaptive responses toneutralize oxidative stress. The organ most highlyadaptive to oxidative damage was liver, compared togill and kidney. Furthermore, the tissue-specific anti-oxidant molecules inhibition/induction proves to be apotent cause of phagocyte-induced peroxidative damageand may be helpful in assessing the oxidative damagerisk associated with the water quality.

Acknowledgments

The financial support from the FCT (Government ofPortugal) provided through Contract S.F.R.H/B.P.D/

ARTICLE IN PRESSI. Ahmad et al. / Ecotoxicology and Environmental Safety 57 (2004) 290–302 301

3603/2000 and by the Aveiro University ResearchInstitute/CESAM is gratefully acknowledged.

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