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Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil
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Ecotoxicology and Environmental Safety 62 (2005) 376–382 www.elsevier.com/locate/ecoenv
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Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil Angela Zaccaron da Silvaa, Juliano Zanettea, Jaime Fernando Ferreirab, Joa˜o Guzenskic, Maria Risoleta Freire Marquesa, Afonso Celso Dias Bainya, Laborato´rio de Biomarcadores de Contaminac- a˜o Aqua´tica e Imunoquı´mica, Departamento de Bioquı´mica, CCB, Universidade Federal de Santa Catarina, 88040-900, Floriano´polis, SC, Brazil b Laborato´rio de Moluscos Marinhos, Departamento de Aquicultura, CCA, Universidade Federal de Santa Catarina, 88040-900, Floriano´polis, SC, Brazil c Empresa de Pesquisa Agropecua´ria e Extensa˜o Rural de Santa Catarina, 88034-901, Floriano´polis, SC, Brazil
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Received 8 October 2004; received in revised form 10 December 2004; accepted 10 December 2004 Available online 25 January 2005
Abstract Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35 ppt) on the activities of glutathione Stransferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1 ml L1 and after depuration for 24 h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15 ppt and remained slightly elevated even after depuration periods of 24 h and 7 days. No changes were observed in the activities of G6PDH, CAT, and AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25 ppt, values usually observed in mangrove ecosystems. r 2005 Elsevier Inc. All rights reserved. Keywords: Mangrove oyster; Crassostrea rhizophorae; Diesel; Salinity; Glutathione S-transferase; Biomarkers
1. Introduction Coastal regions are frequently impacted by human exploration of natural resources. Many foreign organic and inorganic compounds may enter the marine environment through discharge of domestic sewage and industrial effluents, harbor activities, and application of biocides, deteriorating the quality of the aquatic environment. Polycyclic aromatic hydrocarbons (PAHs) are present in the marine environment due to their widespread occurrence in petroleum, coal, soot, air pollutants, and oil spillages (Walker et al., 1996), and Corresponding author. Fax: +55 48 331 9672.
E-mail address: [email protected] (A.C.D. Bainy). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.12.008
carcinogenic properties have been associated with the exposure to these compounds (Reynaud et al., 2002). The impact of these contaminants in biological systems can be evaluated through biomarkers related to xenobiotic biotransformation and excretion mechanisms. Biotransformation of xenobiotics may enhance the production of reactive oxygen species (ROS) and electrophilic intermediates derived from the parent chemical. These intermediates can be conjugated with endogenous molecules such as reduced glutathione (GSH) through the action of glutatione S-transferase (GST) (Livingstone, 1985). This conjugation mechanism produces more hydrophilic xenobiotics yielding expellable metabolites (Mannervik and Danielson, 1988).
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Cellular antioxidant defenses, such as the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, and free radical scavengers, such as vitamins C and E, carotenoids, and GSH among others (Stegeman et al., 1992), protect the cell against oxidative stress, inactivating the produced ROS and/or repairing oxidized biomolecules. Glucose-6-phosphate dehydrogenase (G6PDH) is a regulatory enzyme of the pentose phosphate shunt, which produces NADPH for xenobiotic biotransformation and to recycle oxidized glutathione (GSSG) to its reduced form (GSH) through a reaction catalyzed by glutathione reductase (GR). Acetylcholinesterase (AChE) is a serine hydrolase found in neuromuscular junctions that decomposes acetylcholine in the synaptic cleft (Galloway et al., 2002). AChE activity has been widely used as a biomarker of exposure to and effects of organophosphates and carbamates in aquatic organisms. Payne et al. (1996) showed that AChE is inhibited by aromatic hydrocarbons and suggested the use of the activity of this enzyme to monitor the exposure of aquatic organisms to these compounds. The International Council for the Exploration of the Sea (ICES) has proposed the use of antioxidant and biotransformation enzymes and the inhibition of AChE as biomarkers of exposure to xenobiotics. These parameters have been studied in mussels (Livingstone et al., 1985; Le Pennec and Le Pennec, 2003) and oysters (Alves et al., 2002; Niyogi et al., 2001a). Bivalves are sessile, filter-feeding organisms found in coastal and estuarine zones, which bioaccumulate chemical compounds present in the surrounding seawater; this is the reason that they are used as sentinel organisms in biomonitoring programs (Viarengo and Canesi, 1991; Bainy et al., 2000; Cheung et al., 2001; Oliver et al., 2001; Gowland et al., 2002). The mangrove oyster Crassostrea rhizophorae is a euryhaline osmo-conformer bivalve widely distributed along the Brazilian coast that has been proposed as a relevant biomonitor of environmental contamination in tropical systems (Nascimento et al., 1998; WallnerKersanach et al., 2000; Monserrat et al., 2002; Rebelo et al., 2003). They are found attached to mangrove roots and coastal rocks (Queiroz and Ju´nior, 1990) where drastic changes of salinity may occur, determining their distribution and affecting their structural and functional properties (Dame, 1996). Therefore, examining the effects of salinity on the toxicity of water-borne contaminants is essential in assessing the risk of exposure of estuarine organisms to these compounds (Wang et al., 2001). The aim of this study was to assess the effects of salinity on the activities of GST, G6PDH, CAT, and AChE in digestive glands of C. rhizophorae exposed to diesel oil.
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2. Material and methods 2.1. Animals Mangrove oysters, C. rhizophorae (average length between 60 and 80 mm), were sampled at an oyster farming area at Sambaqui beach (Laborato´rio de Moluscos Marinhos, Departamento de Aquicultura, CCA, UFSC) in Floriano´polis, Santa Catarina State, Brazil. The animals were collected, cleaned, and transported to the laboratory where the experiments were carried out. No sex identification was performed for these organisms. 2.2. Experimental exposure of the oysters The oysters were divided into 16 20-L aquaria, containing filtered seawater disinfected through UV treatment, with constant aeration and temperature at 2372 1C (mean7SD). The animals were divided into four groups of 4 aquaria. Each aquarium contained initially 20 oysters. Each group was gradually acclimated to the experimental salinities of 35, 25, 15, and 9 ppt during a period of 16 days. After the acclimation period, 5 oysters from each salinity group were killed for analysis. The remaining oysters, kept in salinities of 35, 25, 15, and 9 ppt, were exposed to diesel oil at nominal concentrations of 0.01, 0.1, and 1 ml L1 for 7 days. The diesel oil was bought at a commercial gas station from Petroleo Brasileiro S.A., with a certified quality control. The diesel oil was directly added to water and mixed throughout for 5 min. The oysters were immediately placed in the aquarium and remained immersed during the whole period of exposure. Control groups, without diesel addition, were set up in all salinity groups. After the 7-day exposure period, the oysters exposed to increasing diesel oil concentrations, at different salinities, were killed for biochemical analysis. Other groups of oysters that were exposed to diesel were transferred to aquaria containing clean water at different salinities for a 24-h and a 7-day depuration period, when the organisms were subsequently killed for analysis. During the acclimation, exposure, and depuration periods, the oysters were fed with Isochrysis sp. and Chaetoceros muelleri at a density of 1 105 cells per oyster twice a day and the water was renewed daily. 2.3. Sample preparation and biochemical assays At all sampling periods, the digestive gland from each organism was dissected out and frozen individually in liquid nitrogen. The tissues were homogenized (1:4 v/v) with a tissue tearor (Biospec Prod. Inc.) in buffer (20 mM Tris–HCl, pH 7.6, containing 1 mM EDTA, 0.5 M sucrose, 1 mM dithiothrectol, 0.15 M KCl, and 0.1 M phenylmethylsulfonyl fluoride). The homogenate
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was centrifuged at 9000g at 4 1C for 30 min. The supernatant fractions were stored at 80 1C. Glutathione S-transferase (EC 2.5.1.18) activity was estimated as described by Habig and Jakoby (1981), adapted to a 96-well microplate reader (Tecan Sunrise). The assay was performed using 1-chloro-2,4-dinitrobenzene (1 mM) and reduced glutathione (1 mM) in potassium phosphate buffer (0.1 M), pH 7.0. The activity was monitored for 2 min at 340 nm. Glucose 6phosphate dehydrogenase (EC 1.11.49) activity was measured according to Glock and McLean (1953) with adaptations to a 96-well microplate reader. The method is based on the transformation of glucose 6-phosphate by the enzyme through the reduction of NADP+ into NADPH, which is monitored for 2 min at 340 nm. Catalase (EC 1.11.1.6) activity was measured as described by Beutler (1975), but the hydrogen peroxide decomposition was monitored at 240 nm for 1 min. Acetylcholinesterase (EC 3.1.1.7) activity was determined according to Ellman et al. (1961), using 5,50 dithiobis-2-nitrobenzoate (0.01 M, pH 8.0) and acetylthiocholine (0.3 mM) as previously adapted by Monserrat et al. (2002). The reaction was monitored at 412 nm for 2 min at 25 1C. The protein content was determined according to Peterson (1977), using bovine serum albumin as standard.
2.4. Statistical analysis A normal probability test was used to test the data normality. The influences of salinity and diesel oil
exposure on the biochemical parameters were evaluated using two-way analyses of variance (Po0:05), followed by multiple average comparisons according to the Tukey test, for unequal sample sizes (Zar, 1984). StatSoft Statistica 5.1 was used to perform the tests.
3. Results Oyster mortality was observed only in the group exposed to the highest diesel oil concentration (1 ml L1). Four oysters kept at salinity 35 ppt died during the oil exposure, two died during the 24-h depuration period, and three died during the 7-day depuration period. At salinity 15 ppt, one oyster died during the exposure period. No mortality was observed in the oysters exposed to diesel oil and further depurated in salinities 9 and 25 ppt. Fig. 1 shows the GST activity in the digestive gland of oysters sampled after the acclimation, exposure, and depuration periods, at different salinities. The control oysters from the diesel exposure groups at salinities 25 and 15 ppt that were killed after 7 days showed significantly lower GST activity than the oysters analyzed after the acclimation period of 16 days. The control oysters kept at salinity 9 ppt and killed after the exposure period showed higher GST activity than the oysters kept at higher salinities (Fig. 1). The oysters exposed to increasing diesel oil concentrations that were kept at salinities 15 and 25 ppt showed significant increases in GST activity compared to that of the
Fig. 1. Specific activity of GST at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation. *Indicates statistical difference (Po0:05) between the groups. #Indicates statistical difference between the control group kept at alinity 9 ppt and the control groups kept at salinity 35, 25, and 15, after the exposure period.
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Fig. 2. Specific activity of G6PDH at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
Fig. 3. Specific activity of CAT at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
controls (Fig. 1). After both 24-h and 7-day depuration periods, the GST activity in the oysters kept at salinities 15 and 25 ppt were still elevated but not statistically different from that of the controls (Fig. 1). GST activity of oysters exposed to increasing diesel oil concentrations, which were kept at salinities 35 and 9 ppt, was not statistically different from that of the controls (Fig. 1). Activities of G6PDH, CAT, and AChE in the digestive glands of oysters exposed to the various diesel concentrations (0.01, 0.1, and 1 ml L1) in all tested salinities were not different from those of the controls (P40:05) (Figs. 2–4). Likewise, no changes in the
activities of G6PDH, CAT, and AChE were observed in the organisms analyzed after both periods of depuration at different salinities (Figs. 2–4).
4. Discussion The influence of salinity in biomarker responses is especially relevant in euryhaline mollusks, such as C. rhizophorae, which has been proposed as sentinel organism in biomonitoring programs in mangrove and estuarine zones (Nascimento et al., 1998;
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Fig. 4. Specific activity of AChE at salinities 35, 25, 15. and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
Wallner-Kersanach et al., 2000; Monserrat et al., 2002; Rebelo et al., 2003). Frequently these areas receive contaminant inputs associated to domestic and industrial effluent discharges and elevated stormwater runoff. In addition, these factors under low tides can significantly contribute to decrease the salinity where the organisms live. Field studies have shown that the concentrations of high-molecular-weight PAHs were related to the increase in GST activity in the digestive gland of the mussel Perna viridis (Cheung et al., 2001) and in Mytilus edulis (Gowland et al., 2002). In an in vitro model, Le Pennec and Le Pennec (2003) observed that cells from the digestive gland of Pecten maximus, incubated with PAHs for 96 h, showed significant increases in GST activity up to 48 h upon exposure. Longer periods of incubation were followed by lower enzymatic activity and elevated cell death compared to the nontreated cells. In the present study, oysters acclimated to salinities of 25 and 15 ppt and exposed to different concentrations of diesel for 7 days showed concentration-dependent increases in GST activity. This effect was not seen in the oysters acclimated and kept at salinities of 35 and 9 ppt. According to Castro et al. (1985), C. rhizophorae shows higher filtration rates at salinities 20 and 25 ppt, with values of 1.33 and 1.43 L/h, respectively, at temperature of 24 1C. Lower filtration rates of 0.85 and 0.4 L/h were observed at salinities 10 and 35 ppt, respectively. Based on this study, we could suggest that the oysters acclimated to salinities 25 and 15 ppt displayed higher filtration rates. An increase in the energy availability supplied by feeding could provide adequate conditions for efficient xenobiotic conjugation
reactions. It is worth pointing out that the oysters were fed with microalgae daily throughout the experiment. On the other hand, we cannot discard the possibility that higher filtration rates, observed at salinity 25 ppt, imply higher PAH exposure, possibly contributing to the observed elevation in the GST activity. PAH analysis of the tissues of the exposed oysters could test this hypothesis; however, due to technical problems we were unable to perform such an analysis. The elevation of GST activity in the diesel-oil-exposed oysters could also be related to its function in the defense against oxidative stress (Fiander and Schneider, 1999; Amicarelli et al., 2004), reducing organic hydroperoxides possibly formed during the biotransformation of diesel oil compounds. This is consistent with the fact that no changes in the levels of lipid peroxidation were observed in the oysters acclimated to 25 ppt and exposed to diesel oil, but higher levels of this parameter were observed in the organisms exposed at salinities 9, 15, and 35 ppt (J. Zanette, unpublished data). The lack of GST response in the digestive gland of oysters exposed to diesel oil that were kept at salinities of 35 and 9 ppt might be attributed to a greater energetic cost for these organisms to adapt to hyper- and hypoosmotic conditions of the external environment, respectively. Another possibility would be lower contaminant uptake, since in these salinities the filtration rates might have been decreased. According to Dame (1996) and Gosling (2003), bivalves may immediately close their valves when the surrounding salinity is changed as a strategy of protection against osmotic stress. This behavior has been observed by different authors who demonstrated that at low salinities animals
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such as the brown mussel Perna perna (Saloma˜o et al., 1980) and the tropical clam Anomalocardia brasiliana (Leonel et al., 1983) are able to isolate themselves from the surrounding media using this mechanism. Based on these data, we could suggest that PAHs availability to the oysters could be affected by valve closure. Moreover, it is plausible to consider that the absence of a significant effect in the GST response in diesel-oil-exposed oysters that were maintained at 9 ppt could be also associated to the elevated levels of GST activity observed in the control oysters kept in this salinity. The etiology of this response remains to be clarified. During the experiment, death was observed in the oysters exposed to 1 ml L1 and kept at salinity 35 ppt. This observation could be associated with a decreased capacity to conjugate xenobiotics in these animals, which would lead to a rise in the levels of intracellular toxic metabolites and disrupt the homeostasis of the organisms. No significant changes in the G6PDH activity in the digestive gland of the oysters exposed to diesel oil for 7 days at different salinities were seen. G6PDH is the main regulatory enzyme in the pentose phosphate shunt which produces NAPDH, required for the maintenance of the cellular antioxidant capacity (Sies, 1993), fatty acid biosynthesis (Bayne, 1976), and biotransformation of xenobiotics (Timbrel, 1991). NADPH donates electrons to reduce GSSG through the enzyme GR, recycling GSH, required for the GST activity. Considering that no changes in the G6PDH activity in the digestive gland were seen, we could hypothesize that the levels of cellular NAPDH were not affected during the exposure period at different salinities. In fact, the increases in the GST activity at salinities 25 and 15 ppt suggest that the GSH availability was not limited during the exposure. However, when the oysters were exposed to diesel at salinities 35 and 9 ppt, a lack of GST response was observed. This result could be related to lower GR activity, which could decrease GSH recycling capacity. However, this enzyme was not analyzed in the present study. Previous studies carried out in the liver of tilapia (Oreochromis niloticus) caught at a site contaminated by domestic and industrial effluents showed lower activities of both G6PDH and GR together with a smaller GSH content compared to those of noncontaminated reference fish (Bainy et al., 1996). CAT activity in the digestive gland of the oysters was not significantly different in any diesel-exposed group compared to that of controls. Considering that CAT is an important antioxidant enzyme that decomposes hydrogen peroxide that is produced in larger amounts during the biotransformation process, we could suggest that this antioxidant system was not affected by exposure to diesel, at least in the tested concentrations. However, this assumption must be made with caution since some studies have shown positive relationships
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between CAT activity and PAH levels in the digestive gland of the oyster Saccostrea cucullata (Niyogi et al., 2001a), in the digestive tissue of the barnacle Balanus balanoides (Niyogi et al., 2001b), and in the gills of the mussel P. viridis (Cheung et al., 2001). Cheung et al. (2001) observed an inverse correlation between CAT and hydrocarbons in the digestive glands of the same animals. Krishnakumar et al. (1997) did not observe correlations between CAT activity and PAHs in digestive glands of M. edulis exposed to microencapsulated PAHs for periods of 6 and 30 days. These apparent conflicting results could be partially explained by either species- or tissue-specific factors or by abiotic factors, such as the period of exposure, the method of analysis, and the PAH composition. AChE inhibition has been extensively used as a biomarker of exposure to and effects caused by pesticides, especially organophosphates and carbamates (Bocquene´ et al., 1997). Some authors have observed AChE inhibition in mussels caused by exposure to heavy metals (Najimi et al., 1997), and domestic effluents and petroleum (Payne et al., 1996). El-Alfy and Schlenk (1998) observed the inhibition of AChE activity in the euryhaline fish Oryzias latipes exposed to aldicarb and kept at higher salinities. In the present study, neither a significant inhibitory response nor an elevation in the AChE activity was seen upon exposure to diesel at different salinities. This observation indicates that the cholinergic transmission system in the digestive gland of this species was not affected by the diesel exposure and that AChE does not seem to be an adequate biomarker for this purpose. After the 24-h and 7-day depuration periods, the 1ml L1 diesel-exposed oysters kept at salinities 25 and 15 ppt showed decreases in GST activity compared to the groups exposed for 7 days to 1 ml L1 diesel at the same salinities (Fig. 1). This decrease might be associated with lower levels of intracellular toxic metabolites due to an efficient excretion of conjugates during depuration. In addition to the conjugation systems, it is possible that the monoxygenases were also activated in the oysters during the diesel exposure and depuration periods. A similar study showed elevated cytochrome P450 content in the mussel M. edulis exposed to diesel for 4 months, but after 8 days of depuration the expression of these proteins was not different from that of the controls (Livingstone et al., 1985). WallnerKersanach et al. (2000) showed that the oyster C. rhizophorae, exposed for 60 days to a site contaminated by trace metals and depurated for 30 days, eliminated 35% of absorbed lead. In conclusion, the observed dose-dependent increases in GST activity in oysters exposed to diesel at salinities 15 and 25 ppt, followed by decreases in GST activity after depuration, suggest that GST activity can be used
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as a biomarker of exposure to diesel in mangrove and estuarine areas. The lack of changes in the activities of G6PDH, CAT, and AChE in this study support the idea that the activities of these enzymes in the digestive gland of the oyster C. rhizophorae are not suitable biomarkers of an acute exposure to diesel at the concentrations tested. It is possible that additional studies using different exposure periods and diesel oil concentrations would produce different results.
Acknowledgments This work was supported by grants from Brazilian Agency CNPq-CTPetro No. 474513/01-7 and Plano Sul de Pesquisa e Po´s-Graduac- a˜o No. 520741/99-4. A.B. is a research fellow from CNPq. The authors thank Dr. Paulo S.M. Carvalho for revising the manuscript and the two anonymous referees for the excellent suggestions to improve the quality of the manuscript.
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Effects of salinity on biomarker responses in Crassostrea rhizophorae (Mollusca, Bivalvia) exposed to diesel oil Angela Zaccaron da Silvaa, Juliano Zanettea, Jaime Fernando Ferreirab, Joa˜o Guzenskic, Maria Risoleta Freire Marquesa, Afonso Celso Dias Bainya, Laborato´rio de Biomarcadores de Contaminac- a˜o Aqua´tica e Imunoquı´mica, Departamento de Bioquı´mica, CCB, Universidade Federal de Santa Catarina, 88040-900, Floriano´polis, SC, Brazil b Laborato´rio de Moluscos Marinhos, Departamento de Aquicultura, CCA, Universidade Federal de Santa Catarina, 88040-900, Floriano´polis, SC, Brazil c Empresa de Pesquisa Agropecua´ria e Extensa˜o Rural de Santa Catarina, 88034-901, Floriano´polis, SC, Brazil
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Received 8 October 2004; received in revised form 10 December 2004; accepted 10 December 2004 Available online 25 January 2005
Abstract Crassostrea rhizophorae is a euryhaline oyster that inhabits mangrove areas, which are widely distributed along the Brazilian coast. The aim of this study was to investigate the effects of salinity (9, 15, 25, and 35 ppt) on the activities of glutathione Stransferase (GST), glucose 6-phosphate dehydrogenase (G6PDH), catalase (CAT), and acetylcholinesterase (AChE) in the digestive gland of this species after exposure to diesel oil for 7 days at nominal concentrations of 0.01, 0.1, and 1 ml L1 and after depuration for 24 h and 7 days. GST activity increased in a diesel oil concentration-dependent manner at salinities 25 and 15 ppt and remained slightly elevated even after depuration periods of 24 h and 7 days. No changes were observed in the activities of G6PDH, CAT, and AChE in the oysters exposed to diesel and depurated. Based on these results, GST activity in the digestive gland of C. rhizophorae might be used as a biomarker of exposure to diesel oil in sites where the salinity is between 15 and 25 ppt, values usually observed in mangrove ecosystems. r 2005 Elsevier Inc. All rights reserved. Keywords: Mangrove oyster; Crassostrea rhizophorae; Diesel; Salinity; Glutathione S-transferase; Biomarkers
1. Introduction Coastal regions are frequently impacted by human exploration of natural resources. Many foreign organic and inorganic compounds may enter the marine environment through discharge of domestic sewage and industrial effluents, harbor activities, and application of biocides, deteriorating the quality of the aquatic environment. Polycyclic aromatic hydrocarbons (PAHs) are present in the marine environment due to their widespread occurrence in petroleum, coal, soot, air pollutants, and oil spillages (Walker et al., 1996), and Corresponding author. Fax: +55 48 331 9672.
E-mail address: [email protected] (A.C.D. Bainy). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.12.008
carcinogenic properties have been associated with the exposure to these compounds (Reynaud et al., 2002). The impact of these contaminants in biological systems can be evaluated through biomarkers related to xenobiotic biotransformation and excretion mechanisms. Biotransformation of xenobiotics may enhance the production of reactive oxygen species (ROS) and electrophilic intermediates derived from the parent chemical. These intermediates can be conjugated with endogenous molecules such as reduced glutathione (GSH) through the action of glutatione S-transferase (GST) (Livingstone, 1985). This conjugation mechanism produces more hydrophilic xenobiotics yielding expellable metabolites (Mannervik and Danielson, 1988).
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Cellular antioxidant defenses, such as the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, and free radical scavengers, such as vitamins C and E, carotenoids, and GSH among others (Stegeman et al., 1992), protect the cell against oxidative stress, inactivating the produced ROS and/or repairing oxidized biomolecules. Glucose-6-phosphate dehydrogenase (G6PDH) is a regulatory enzyme of the pentose phosphate shunt, which produces NADPH for xenobiotic biotransformation and to recycle oxidized glutathione (GSSG) to its reduced form (GSH) through a reaction catalyzed by glutathione reductase (GR). Acetylcholinesterase (AChE) is a serine hydrolase found in neuromuscular junctions that decomposes acetylcholine in the synaptic cleft (Galloway et al., 2002). AChE activity has been widely used as a biomarker of exposure to and effects of organophosphates and carbamates in aquatic organisms. Payne et al. (1996) showed that AChE is inhibited by aromatic hydrocarbons and suggested the use of the activity of this enzyme to monitor the exposure of aquatic organisms to these compounds. The International Council for the Exploration of the Sea (ICES) has proposed the use of antioxidant and biotransformation enzymes and the inhibition of AChE as biomarkers of exposure to xenobiotics. These parameters have been studied in mussels (Livingstone et al., 1985; Le Pennec and Le Pennec, 2003) and oysters (Alves et al., 2002; Niyogi et al., 2001a). Bivalves are sessile, filter-feeding organisms found in coastal and estuarine zones, which bioaccumulate chemical compounds present in the surrounding seawater; this is the reason that they are used as sentinel organisms in biomonitoring programs (Viarengo and Canesi, 1991; Bainy et al., 2000; Cheung et al., 2001; Oliver et al., 2001; Gowland et al., 2002). The mangrove oyster Crassostrea rhizophorae is a euryhaline osmo-conformer bivalve widely distributed along the Brazilian coast that has been proposed as a relevant biomonitor of environmental contamination in tropical systems (Nascimento et al., 1998; WallnerKersanach et al., 2000; Monserrat et al., 2002; Rebelo et al., 2003). They are found attached to mangrove roots and coastal rocks (Queiroz and Ju´nior, 1990) where drastic changes of salinity may occur, determining their distribution and affecting their structural and functional properties (Dame, 1996). Therefore, examining the effects of salinity on the toxicity of water-borne contaminants is essential in assessing the risk of exposure of estuarine organisms to these compounds (Wang et al., 2001). The aim of this study was to assess the effects of salinity on the activities of GST, G6PDH, CAT, and AChE in digestive glands of C. rhizophorae exposed to diesel oil.
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2. Material and methods 2.1. Animals Mangrove oysters, C. rhizophorae (average length between 60 and 80 mm), were sampled at an oyster farming area at Sambaqui beach (Laborato´rio de Moluscos Marinhos, Departamento de Aquicultura, CCA, UFSC) in Floriano´polis, Santa Catarina State, Brazil. The animals were collected, cleaned, and transported to the laboratory where the experiments were carried out. No sex identification was performed for these organisms. 2.2. Experimental exposure of the oysters The oysters were divided into 16 20-L aquaria, containing filtered seawater disinfected through UV treatment, with constant aeration and temperature at 2372 1C (mean7SD). The animals were divided into four groups of 4 aquaria. Each aquarium contained initially 20 oysters. Each group was gradually acclimated to the experimental salinities of 35, 25, 15, and 9 ppt during a period of 16 days. After the acclimation period, 5 oysters from each salinity group were killed for analysis. The remaining oysters, kept in salinities of 35, 25, 15, and 9 ppt, were exposed to diesel oil at nominal concentrations of 0.01, 0.1, and 1 ml L1 for 7 days. The diesel oil was bought at a commercial gas station from Petroleo Brasileiro S.A., with a certified quality control. The diesel oil was directly added to water and mixed throughout for 5 min. The oysters were immediately placed in the aquarium and remained immersed during the whole period of exposure. Control groups, without diesel addition, were set up in all salinity groups. After the 7-day exposure period, the oysters exposed to increasing diesel oil concentrations, at different salinities, were killed for biochemical analysis. Other groups of oysters that were exposed to diesel were transferred to aquaria containing clean water at different salinities for a 24-h and a 7-day depuration period, when the organisms were subsequently killed for analysis. During the acclimation, exposure, and depuration periods, the oysters were fed with Isochrysis sp. and Chaetoceros muelleri at a density of 1 105 cells per oyster twice a day and the water was renewed daily. 2.3. Sample preparation and biochemical assays At all sampling periods, the digestive gland from each organism was dissected out and frozen individually in liquid nitrogen. The tissues were homogenized (1:4 v/v) with a tissue tearor (Biospec Prod. Inc.) in buffer (20 mM Tris–HCl, pH 7.6, containing 1 mM EDTA, 0.5 M sucrose, 1 mM dithiothrectol, 0.15 M KCl, and 0.1 M phenylmethylsulfonyl fluoride). The homogenate
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was centrifuged at 9000g at 4 1C for 30 min. The supernatant fractions were stored at 80 1C. Glutathione S-transferase (EC 2.5.1.18) activity was estimated as described by Habig and Jakoby (1981), adapted to a 96-well microplate reader (Tecan Sunrise). The assay was performed using 1-chloro-2,4-dinitrobenzene (1 mM) and reduced glutathione (1 mM) in potassium phosphate buffer (0.1 M), pH 7.0. The activity was monitored for 2 min at 340 nm. Glucose 6phosphate dehydrogenase (EC 1.11.49) activity was measured according to Glock and McLean (1953) with adaptations to a 96-well microplate reader. The method is based on the transformation of glucose 6-phosphate by the enzyme through the reduction of NADP+ into NADPH, which is monitored for 2 min at 340 nm. Catalase (EC 1.11.1.6) activity was measured as described by Beutler (1975), but the hydrogen peroxide decomposition was monitored at 240 nm for 1 min. Acetylcholinesterase (EC 3.1.1.7) activity was determined according to Ellman et al. (1961), using 5,50 dithiobis-2-nitrobenzoate (0.01 M, pH 8.0) and acetylthiocholine (0.3 mM) as previously adapted by Monserrat et al. (2002). The reaction was monitored at 412 nm for 2 min at 25 1C. The protein content was determined according to Peterson (1977), using bovine serum albumin as standard.
2.4. Statistical analysis A normal probability test was used to test the data normality. The influences of salinity and diesel oil
exposure on the biochemical parameters were evaluated using two-way analyses of variance (Po0:05), followed by multiple average comparisons according to the Tukey test, for unequal sample sizes (Zar, 1984). StatSoft Statistica 5.1 was used to perform the tests.
3. Results Oyster mortality was observed only in the group exposed to the highest diesel oil concentration (1 ml L1). Four oysters kept at salinity 35 ppt died during the oil exposure, two died during the 24-h depuration period, and three died during the 7-day depuration period. At salinity 15 ppt, one oyster died during the exposure period. No mortality was observed in the oysters exposed to diesel oil and further depurated in salinities 9 and 25 ppt. Fig. 1 shows the GST activity in the digestive gland of oysters sampled after the acclimation, exposure, and depuration periods, at different salinities. The control oysters from the diesel exposure groups at salinities 25 and 15 ppt that were killed after 7 days showed significantly lower GST activity than the oysters analyzed after the acclimation period of 16 days. The control oysters kept at salinity 9 ppt and killed after the exposure period showed higher GST activity than the oysters kept at higher salinities (Fig. 1). The oysters exposed to increasing diesel oil concentrations that were kept at salinities 15 and 25 ppt showed significant increases in GST activity compared to that of the
Fig. 1. Specific activity of GST at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation. *Indicates statistical difference (Po0:05) between the groups. #Indicates statistical difference between the control group kept at alinity 9 ppt and the control groups kept at salinity 35, 25, and 15, after the exposure period.
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Fig. 2. Specific activity of G6PDH at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
Fig. 3. Specific activity of CAT at salinities 35, 25, 15, and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
controls (Fig. 1). After both 24-h and 7-day depuration periods, the GST activity in the oysters kept at salinities 15 and 25 ppt were still elevated but not statistically different from that of the controls (Fig. 1). GST activity of oysters exposed to increasing diesel oil concentrations, which were kept at salinities 35 and 9 ppt, was not statistically different from that of the controls (Fig. 1). Activities of G6PDH, CAT, and AChE in the digestive glands of oysters exposed to the various diesel concentrations (0.01, 0.1, and 1 ml L1) in all tested salinities were not different from those of the controls (P40:05) (Figs. 2–4). Likewise, no changes in the
activities of G6PDH, CAT, and AChE were observed in the organisms analyzed after both periods of depuration at different salinities (Figs. 2–4).
4. Discussion The influence of salinity in biomarker responses is especially relevant in euryhaline mollusks, such as C. rhizophorae, which has been proposed as sentinel organism in biomonitoring programs in mangrove and estuarine zones (Nascimento et al., 1998;
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Fig. 4. Specific activity of AChE at salinities 35, 25, 15. and 9 ppt. Each bar indicates nominal diesel concentration used during the exposure period and numbers of individuals are given between brackets. Data are presented as mean7standard deviation.
Wallner-Kersanach et al., 2000; Monserrat et al., 2002; Rebelo et al., 2003). Frequently these areas receive contaminant inputs associated to domestic and industrial effluent discharges and elevated stormwater runoff. In addition, these factors under low tides can significantly contribute to decrease the salinity where the organisms live. Field studies have shown that the concentrations of high-molecular-weight PAHs were related to the increase in GST activity in the digestive gland of the mussel Perna viridis (Cheung et al., 2001) and in Mytilus edulis (Gowland et al., 2002). In an in vitro model, Le Pennec and Le Pennec (2003) observed that cells from the digestive gland of Pecten maximus, incubated with PAHs for 96 h, showed significant increases in GST activity up to 48 h upon exposure. Longer periods of incubation were followed by lower enzymatic activity and elevated cell death compared to the nontreated cells. In the present study, oysters acclimated to salinities of 25 and 15 ppt and exposed to different concentrations of diesel for 7 days showed concentration-dependent increases in GST activity. This effect was not seen in the oysters acclimated and kept at salinities of 35 and 9 ppt. According to Castro et al. (1985), C. rhizophorae shows higher filtration rates at salinities 20 and 25 ppt, with values of 1.33 and 1.43 L/h, respectively, at temperature of 24 1C. Lower filtration rates of 0.85 and 0.4 L/h were observed at salinities 10 and 35 ppt, respectively. Based on this study, we could suggest that the oysters acclimated to salinities 25 and 15 ppt displayed higher filtration rates. An increase in the energy availability supplied by feeding could provide adequate conditions for efficient xenobiotic conjugation
reactions. It is worth pointing out that the oysters were fed with microalgae daily throughout the experiment. On the other hand, we cannot discard the possibility that higher filtration rates, observed at salinity 25 ppt, imply higher PAH exposure, possibly contributing to the observed elevation in the GST activity. PAH analysis of the tissues of the exposed oysters could test this hypothesis; however, due to technical problems we were unable to perform such an analysis. The elevation of GST activity in the diesel-oil-exposed oysters could also be related to its function in the defense against oxidative stress (Fiander and Schneider, 1999; Amicarelli et al., 2004), reducing organic hydroperoxides possibly formed during the biotransformation of diesel oil compounds. This is consistent with the fact that no changes in the levels of lipid peroxidation were observed in the oysters acclimated to 25 ppt and exposed to diesel oil, but higher levels of this parameter were observed in the organisms exposed at salinities 9, 15, and 35 ppt (J. Zanette, unpublished data). The lack of GST response in the digestive gland of oysters exposed to diesel oil that were kept at salinities of 35 and 9 ppt might be attributed to a greater energetic cost for these organisms to adapt to hyper- and hypoosmotic conditions of the external environment, respectively. Another possibility would be lower contaminant uptake, since in these salinities the filtration rates might have been decreased. According to Dame (1996) and Gosling (2003), bivalves may immediately close their valves when the surrounding salinity is changed as a strategy of protection against osmotic stress. This behavior has been observed by different authors who demonstrated that at low salinities animals
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such as the brown mussel Perna perna (Saloma˜o et al., 1980) and the tropical clam Anomalocardia brasiliana (Leonel et al., 1983) are able to isolate themselves from the surrounding media using this mechanism. Based on these data, we could suggest that PAHs availability to the oysters could be affected by valve closure. Moreover, it is plausible to consider that the absence of a significant effect in the GST response in diesel-oil-exposed oysters that were maintained at 9 ppt could be also associated to the elevated levels of GST activity observed in the control oysters kept in this salinity. The etiology of this response remains to be clarified. During the experiment, death was observed in the oysters exposed to 1 ml L1 and kept at salinity 35 ppt. This observation could be associated with a decreased capacity to conjugate xenobiotics in these animals, which would lead to a rise in the levels of intracellular toxic metabolites and disrupt the homeostasis of the organisms. No significant changes in the G6PDH activity in the digestive gland of the oysters exposed to diesel oil for 7 days at different salinities were seen. G6PDH is the main regulatory enzyme in the pentose phosphate shunt which produces NAPDH, required for the maintenance of the cellular antioxidant capacity (Sies, 1993), fatty acid biosynthesis (Bayne, 1976), and biotransformation of xenobiotics (Timbrel, 1991). NADPH donates electrons to reduce GSSG through the enzyme GR, recycling GSH, required for the GST activity. Considering that no changes in the G6PDH activity in the digestive gland were seen, we could hypothesize that the levels of cellular NAPDH were not affected during the exposure period at different salinities. In fact, the increases in the GST activity at salinities 25 and 15 ppt suggest that the GSH availability was not limited during the exposure. However, when the oysters were exposed to diesel at salinities 35 and 9 ppt, a lack of GST response was observed. This result could be related to lower GR activity, which could decrease GSH recycling capacity. However, this enzyme was not analyzed in the present study. Previous studies carried out in the liver of tilapia (Oreochromis niloticus) caught at a site contaminated by domestic and industrial effluents showed lower activities of both G6PDH and GR together with a smaller GSH content compared to those of noncontaminated reference fish (Bainy et al., 1996). CAT activity in the digestive gland of the oysters was not significantly different in any diesel-exposed group compared to that of controls. Considering that CAT is an important antioxidant enzyme that decomposes hydrogen peroxide that is produced in larger amounts during the biotransformation process, we could suggest that this antioxidant system was not affected by exposure to diesel, at least in the tested concentrations. However, this assumption must be made with caution since some studies have shown positive relationships
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between CAT activity and PAH levels in the digestive gland of the oyster Saccostrea cucullata (Niyogi et al., 2001a), in the digestive tissue of the barnacle Balanus balanoides (Niyogi et al., 2001b), and in the gills of the mussel P. viridis (Cheung et al., 2001). Cheung et al. (2001) observed an inverse correlation between CAT and hydrocarbons in the digestive glands of the same animals. Krishnakumar et al. (1997) did not observe correlations between CAT activity and PAHs in digestive glands of M. edulis exposed to microencapsulated PAHs for periods of 6 and 30 days. These apparent conflicting results could be partially explained by either species- or tissue-specific factors or by abiotic factors, such as the period of exposure, the method of analysis, and the PAH composition. AChE inhibition has been extensively used as a biomarker of exposure to and effects caused by pesticides, especially organophosphates and carbamates (Bocquene´ et al., 1997). Some authors have observed AChE inhibition in mussels caused by exposure to heavy metals (Najimi et al., 1997), and domestic effluents and petroleum (Payne et al., 1996). El-Alfy and Schlenk (1998) observed the inhibition of AChE activity in the euryhaline fish Oryzias latipes exposed to aldicarb and kept at higher salinities. In the present study, neither a significant inhibitory response nor an elevation in the AChE activity was seen upon exposure to diesel at different salinities. This observation indicates that the cholinergic transmission system in the digestive gland of this species was not affected by the diesel exposure and that AChE does not seem to be an adequate biomarker for this purpose. After the 24-h and 7-day depuration periods, the 1ml L1 diesel-exposed oysters kept at salinities 25 and 15 ppt showed decreases in GST activity compared to the groups exposed for 7 days to 1 ml L1 diesel at the same salinities (Fig. 1). This decrease might be associated with lower levels of intracellular toxic metabolites due to an efficient excretion of conjugates during depuration. In addition to the conjugation systems, it is possible that the monoxygenases were also activated in the oysters during the diesel exposure and depuration periods. A similar study showed elevated cytochrome P450 content in the mussel M. edulis exposed to diesel for 4 months, but after 8 days of depuration the expression of these proteins was not different from that of the controls (Livingstone et al., 1985). WallnerKersanach et al. (2000) showed that the oyster C. rhizophorae, exposed for 60 days to a site contaminated by trace metals and depurated for 30 days, eliminated 35% of absorbed lead. In conclusion, the observed dose-dependent increases in GST activity in oysters exposed to diesel at salinities 15 and 25 ppt, followed by decreases in GST activity after depuration, suggest that GST activity can be used
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as a biomarker of exposure to diesel in mangrove and estuarine areas. The lack of changes in the activities of G6PDH, CAT, and AChE in this study support the idea that the activities of these enzymes in the digestive gland of the oyster C. rhizophorae are not suitable biomarkers of an acute exposure to diesel at the concentrations tested. It is possible that additional studies using different exposure periods and diesel oil concentrations would produce different results.
Acknowledgments This work was supported by grants from Brazilian Agency CNPq-CTPetro No. 474513/01-7 and Plano Sul de Pesquisa e Po´s-Graduac- a˜o No. 520741/99-4. A.B. is a research fellow from CNPq. The authors thank Dr. Paulo S.M. Carvalho for revising the manuscript and the two anonymous referees for the excellent suggestions to improve the quality of the manuscript.
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