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Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons
Marine Environmental Research 52 (2001) 13±26 www.elsevier.com/locate/marenvrev
Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons S. Niyogi a, S. Biswas a, S. Sarker b, A.G. Datta a,* a Department of Life Science & Biotechnology, Jadavpur University, Calcutta 700032, India Indian Institute of Chemical Biology, 4 Raja S.C. Mallick Road, Jadavpur, Calcutta 700032, India
b
Received 25 October 1999; received in revised form 19 March 2000; accepted 19 July 2000
Abstract Seasonal variations in the antioxidant enzymes (catalase, superoxide dismutase [SOD], NADH-DT diaphorase), biotransformation enzyme, glutathione-S-transferase (GST) and microsomal lipid peroxidation in digestive tissue of barnacle, Balanus balanoides, from polluted and non-polluted populations have been evaluated. Relationships with accumulated polyaromatic hydrocarbon (PAH) concentration in barnacle tissues and environmental parameters (water temperature, salinity, dissolved oxygen concentration, water pH) were determined. As a general trend, maximum antioxidant enzyme and GST activities were detected in the pre-monsoon period or summer (March±June) followed by a gradual decrease during the monsoon (July±October) with a minimum in the post-monsoon period or winter (November± February). This pattern was similar to tissue concentrations of PAHs, resulting in a signi®cant positive correlation with antioxidant enzymes, mainly catalase and SOD. Microsomal lipid peroxidation exhibited an almost reverse trend of seasonal variation to that of antioxidant enzyme activities indicating an enhanced susceptibility of barnacle tissues to oxidative stress. Among the environmental parameters, only water temperature seemed to have a signi®cant eect on observed variations of antioxidant enzymes and GST activities. The barnacles from polluted and non-polluted populations exhibited seasonal dierences in the activities of all the enzymes studied, particularly catalase, SOD and GST, suggesting the possibility of some biochemical adaptation in organisms from a chronically polluted environment. The results indicated that antioxidant defense components, catalase and SOD, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The * Corresponding author. Tel.: +91-33-474-9696. E-mail address: [email protected] (A.G. Datta). 0141-1136/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00257-9
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results also suggested the potentiality of barnacle, B. balanoides, as a bioindicator organism against organic pollution. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hooghly Estuary; Barnacle; Seasonal variation; Physico-chemical parameters; PAHs; Antioxidant enzymes; GST; Biomarkers
1. Introduction Pollution in the marine environment aects not only aquatic life as such but also poses a serious threat to all life on the planet. The rapid advancement of industrial development worldwide has led to a continual in¯ux of xenobiotics into oceans, particularly polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc., which may ultimately upset the fragile balance of life in the seas. Estuaries and coastal waters are particularly at risk from such anthropogenic pollution. It is always very dicult from only contaminant body burden data to obtain information about their signi®cance upon animal health. Therefore, techniques for measuring biological eects are critical for any pollution monitoring program. Among various biochemical systems, monitoring the cytochrome P450 monooxygenase or mixed-function oxygenase (MFO) system is one of the most widely accepted methods for detecting organic pollution in marine environment. The MFO system is an apparently universally distributed enzyme system involved in the detoxi®cation as well as in some cases, activation of xenobiotics such as PAHs (Sato & Omura, 1978). Recently, it has been suggested that antioxidant as well as phase II enzymes like glutathione-S-transferase (GST) in conjunction with MFO system can also be studied to build up a more comprehensive picture of toxic eect and response (Foerlin, Lemaire, Livingstone, Forlin, & Andersson, 1995; Livingstone, 1991; Porte, Sole, Albaiges, & Livingstone, 1991; Regoli, Nigro, Bertoli, Principaro, & Orlando, 1997; Rodriguez-Ariza et al., 1993; Sheehan, McIntosh, Power, & Fitzpatrick, 1994; SoleÂ, Porte, & Albaiges, 1994, 1995a). In this aspect, a majority of the published works are concerned with bivalve molluscs, particularly mussels and oysters, but there is very little information available about these systems in other marine invertebrates like lower order crustaceans, a large, diverse, and complex group of animals. Barnacles (sub class: Cirripedia; Order: Thoracica) (Barnes, 1980) comprise a unique and important group in the class Crustacea. Exclusively marine and having worldwide distribution and great abundance, they are the only group of exclusively sessile, hermaphroditic crustaceans which make them suitable as bioindicator organisms. Although Mitra, Trivedi, Chaudhuri, Bag, Ghosh, and Choudhury (1995) suggested the usefulness of barnacles as an indicator of heavy metal pollution, there is no information available about the potentiality of these organisms for the biomonitoring of organic pollution. Stegeman and Kaplan (1981) characterized cytochrome P450 and its components in acorn barnacle, Balanus eburneus, but the status of antioxidant and other biotransformation enzymes in these organisms and their response to organic pollution is virtually unknown. In this paper, we report the seasonal variations of the activities of three cytosolic antioxidant enzymes [Catalase
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(EC 1.11.1.6), superoxide dismutase SOD EC 1.15.1.1) and DT-diaphorase (EC 1.6.99.2)], a cytosolic phase II enzyme, GST (EC 2.5.1.18) and microsomal lipid peroxidation in the digestive tissue (digestive gland along with intestine) of barnacle, Balanus balanoides, collected from two dierent locations along Hooghly Estuary, India. Seasonal variations of physico-chemical parameters of ambient water (surface water temperature, salinity, pH, dissolved oxygen) and the PAH concentrations in whole-body tissues of barnacles from the two locations were also determined. 2. Site characteristics The Hooghly Estuary (88 000 ±89 280 E longitude and 21 000 ±22 300 N latitude), which supports the world's largest and most magni®cent mangrove block, the Sunderbans, is a typical and very conspicuous ecosystem of the Indian subcontinent. This unique estuary and the Sunderbans are among the world's most precious genetically diverse ecosystems. With the rapid emergence of the Haldia port complex as a major oil disembarkment terminal in eastern India and the industrialization of the surrounding areas, including development of India's largest oil re®nery, the estuary has become vulnerable to pollution, particularly due to petroleum-derived hydrocarbons including PAHs. This Haldia port complex area was chosen as the polluted site of our study (Station 3). The Old Lighthouse Complex at Sagar Island, a remote place inside the mangrove block at the lower stretch of Hooghly Estuary, which is more than 50 km away from the Haldia port complex, was chosen as the reference site (Station 1). Kachubaria, another remote area situated at the northern tip of Sagar Island was also considered for this study (Station 2). Both these areas are less polluted than the Haldia port complex due to the absence of any nearby industrial activity. 3. Methods and materials Barnacles, B. balanoides, were collected from all the three stations (period of study: November 1997±October 1998). Samples of consistent size range (diameter of the base of shell Ð 1.4±1.5 cm) were taken every time as recommended by the guidelines of the Joint Monitoring Group of the Oslo and Paris conventions (Mitra & Choudhury, 1993). Sampling was done twice a month from all the three stations during low tide. The samples were transferred to the laboratory in living condition and processed immediately. Animals were removed from calcareous shells, the digestive tissues (digestive gland with intestine) were dissected out, and ¯uids were rinsed from the intestine. Dissected digestive tissues of 2 g (pooled) were immediately homogenized in 5 vol. of 65 mM Na-phosphate buer, 0.15 M KCl, 3 mM MgCl2, 0.1 mM phenyl methyl sulfonyl ¯uoride, pH 7.0 using a PotterElvehjam-type glass te¯on homogeniser and centrifuged at 12,000g for 30 min at 4 C. The 12,000g supernatant was again centrifuged at 100,000g for 90 min at 4 C. The 100,000g supernatants or cytosol was subsequently used for the
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evaluation of enzyme activities. Catalase activity was assayed by the method of Greenwald (1985). The reaction mixture contained 50 mM phosphate buer (pH 7.0) and 50 mM H2O2. The reaction rate was measured at 240 nm. One unit of catalase activity was de®ned as 1 mmol of H2O2 degraded min 1 g 1 wet weight. SOD activity was determined by the method of Paoletti and Mocali (1990). The inhibitory eect of SOD on the superoxide generation in presence of ethylene-diamine tetra acetic acid (EDTA)±manganese (II) chloride (2.5/1.25 mM), mercaptoethanol (1 mM) and reduced nitcoinamide adenine dinucleotidephosphate (NADPH) (0.3 mM) in triethanolamine-diethanolamine (100 mM each) hydrochloric acid± (HCl) buer (pH 7.4) was analyzed spectrophotometrically at 340 nm. The assays were run without and with dierent volumes of enzyme sample to determine 50% inhibition. One unit of SOD activity was calculated as the amount of enzyme required to inhibit the rate of NADPH oxidation of the control by 50%. Cytosolic nicotinamide adenine dinucleotide hydrogen (NADH-TD) diaphorase activity was measured as dicumarol inhibitable NADH±dichlorophenolindophenol (DCPIP) reductase activity (Livingstone et al., 1990). The reaction mixture contained 50 mM Tris±HCl (pH 7.6), 0.3 mM NADH and 40 mM DCPIP. The decrease in absorbance was monitored at 600 nm with and without 100 mM dicumarol in 0.15% NaOH to determine the actual rate of activity of the enzyme. One unit of NADH-DT diaphorase activity was de®ned as nanomols of DCPIP reduced per minute per milligram protein. Microsomal NADPH-DT diaphorase was measured similarly replacing NADH with NADPH. GST was measured according to Habig, Pabst and Jakoby (1974) using 1-chloro-2-4-dinitrobenzene (CDNB) as substrate. The standard assay mixture contained 0.1 M phosphate buer (pH 6.5), 1 mM reduced glutathione (GSH), 1 mM EDTA and 1 mM CDNB. The complete assay mixture without enzyme was used as a control. The rate of reaction was measured as the increase in absorbance at 340 nm. The chemical rate of reaction, determined in the absence of sample, was subtracted from the sample. One unit of the enzyme was de®ned as 1 nmol of GSH conjugated min 1 mg 1 protein. The 100,000g pellets or microsomes were washed three to four times in 0.1 M phosphate buer, pH 7.3 and ®nally resuspended in 2 ml of the same buer. Lipid peroxidation in microsomal fraction was measured as malondialdehyde equivalent using trichloroacetic acid (TCA), thiobarbituric acid (TBA) and (HCl) reagent (TBA±TCA reagent: 0.375% w/v TBA, 15% w/v TCA and 0.25 N HCl) (Buege & Aust, 1978). Microsomal suspension (1 ml) was mixed with 2 ml of TBA±TCA reagent and heated in a boiling waterbath for 15 min. After cooling, the ¯occulant precipitate was removed by centrifugation at 1000g for 10 min. Finally the malondialdehyde concentration in the supernatant fraction was determined spectrophotometrically at 535 nm using an extinction coecient of 1.56105 M 1 cm 1. The results are expressed as nanomols MDA g 1 wet weight. Proteins in all samples were determined by the method of Bradford (1976) with bovine serum albumin as standard. Chemical analyses of barnacle tissues were carried out, basically according to the method of Porte et al. (1991) with slight modi®cations. The whole soft body tissues of barnacles (1 g) were homogenized and Soxhlet-extracted for 12 h with n-hexane and methylene chloride (4:1). The extract was treated with Na2SO4 overnight to
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remove remaining water and ®nally applied to a silica gel column to purify it. PAH concentration was determined in the corresponding fraction by UV ¯uorescence, emission and excitation intensities being measured at 360 and 310 nm, respectively, and expressed as mg g 1 wet weight (chrysene equivalents). Internal standard (chrysene, 95% pure, procured from Sigma Chemicals Ltd., St Louis, Missouri, USA) was added to tissue homogenate before extraction as a quality control method and the percentage recovery was 88.5 (1.8 S.E.M., n=8). All physico-chemical parameters were assessed during each sampling. Surface water temperature was measured with a Celsius thermometer (ranging 0±100 C). The surface water salinity around the sampling stations were estimated by employing the ``Mohr±Knudssen method'' (Strickland & Parsons, 1968). The correction factor was determined by titrating silver nitrate solution against standard seawater [IAPO standard seawater service, Charlottenlund, Stot, Denmark (salinity 19.376)]. The pH of the ambient media of both stations was determined on the spot by a portable pH meter (sensitivity=0.02 units). Dissolved oxygen in the ambient water was determined by the method as described in Strickland and Parsons. Statistical signi®cance was determined by Student's `t test'. 4. Results Among the physico-chemical parameters (Table 1), salinity exhibited a distinct seasonal pattern in all the three stations. Maximum values were recorded during the pre-monsoon period (March±June), followed by minimum values during the monsoon period (July±October) and again a slight increase during post-monsoon (November±February). Salinity was higher at Station 1 throughout the year followed by Stations 2 and 3, respectively. Other physico-chemical parameters (surface water temperature, pH, dissolved oxygen) exhibited little dierence between the three stations. Surface water temperature was similar during the pre-monsoon and monsoon periods with a moderate decrease during the post-monsoon period. Aquatic pH and the dissolved oxygen decreased slightly during the monsoon period, but otherwise maintained a consistent pattern throughout the year. The PAH concentration in the soft body tissues B. balanoides (Table 2) exhibited high values (six to nine times higher) in animals collected from Station 3 compared to Stations 1 and 2 throughout the year. The PAH concentration in soft body tissues also showed a distinct seasonal pattern with a maxima during the pre-monsoon period followed by gradual decrease during the monsoon to post-monsoon period. All the antioxidant enzymes in the digestive tissues of barnacles at all three stations exhibited a similar seasonal pro®le throughout the year (Fig. 1a, b, c). Maximum activity occurred during the pre-monsoon period with a gradual decrease during monsoon to post-monsoon. The seasonal variation of the biotransformation enzymes, GST, was not as distinct as the antioxidant enzymes (Fig. 2). Although the maxima was observed during the pre-monsoon period, there was very little variation in GST activities between monsoon and post-monsoon periods. Microsomal lipid peroxidation in digestive tissues of barnacles showed a reverse pro®le to that of antioxidant
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Season
Water Temperature ( C)
Salinity (ppt)
Station 1
Station 1
Station 2
Station 3
Station 2
Dissolved oxygen (mg l 1)
pH Station 3
Station 1
Station 2
Station 3
Station 1
Station 2
Station 3
Pre-monsoon 30.70.84 32.20.74 31.10.69 26.90.36 15.20.49 8.340.68 8.280.04 8.200.04 8.190.03 5.890.26 5.410.27 5.620.20 Monsoon 29.90.43 29.60.65 30.10.66 18.11.25 5.40.42 1.50.76 7.970.06 7.980.03 7.880.05 5.280.22 6.020.31 5.150.10 Post-monsoon 24.80.82 25.10.47 24.30.52 22.40.53 9.60.81 2.90.43 8.210.03 8.010.04 8.080.04 5.530.15 5.080.30 5.210.13 a
Values are expressed as meansS.E.M. (n=8).
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Table 1 Seasonal variation of physico-chemical parameters of surface water in three stationsa
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Table 2 Seasonal variation of polyaromatic hydrocarbon concentration in soft body tissue of barnacle, Balanus balanoidesa Season
Station 1
Station 2
Station 3
Pre-monsoon Monsoon Post-monsoon
0.70.08 0.50.07 0.40.06
0.80.10 0.60.06 0.50.06
5.10.71 4.50.87 3.10.22
a
Values are expressed as mg g
1
wet weight (chrysene equivalents) and meansS.E.M. (n=8).
enzymes (Fig. 3). It was lowest during the pre-monsoon period with subsequent increase from the monsoon to post-monsoon period. Our study also revealed that the antioxidant enzymes and GST showed persistent induction in barnacles at the highly contaminated Station 3 compared to Stations 1 and 2. Catalase and SOD maintained signi®cant induction at Station 3 compared to Station 1 (control station) throughout the year. Cytosolic NADH-DT diaphorase exhibited less and insignificant induction. Microsomal NADPH-DT diaphorase could not be detected in barnacles. GST induction was signi®cantly dierent at Station 3 compared to Station 1 during the pre-monsoon period only. Microsomal lipid peroxidation also maintained a consistent elevated level at Station 3 compared to the control station although the dierence was signi®cant only in the post-monsoon period. However, no signi®cant dierence between the activities of any of these enzymes and microsomal lipid peroxidation was observed in barnacles collected from Station 1 and 2. Among the physico-chemical parameters, only surface water temperature showed signi®cant correlation with biochemical parameters (Table 3) whereas PAH concentration in soft body tissue of barnacles correlated signi®cantly with catalase and SOD (Table 4). The linear regression analysis of catalase and SOD on tissue PAH concentration exhibited a positive slope in both cases (Fig. 4a, b). 5. Discussion The results indicate that the seasonal monsoonal cycle in the Indian subcontinent seems to have some eect on physico-chemical parameters of the aquatic environment, particularly on salinity and to some extent on pH. The decrease in salinity and pH in the monsoon period is due to massive rainfall that causes a considerable increase in fresh water run o from the upper stretch of the estuary. The higher salinity at Old Lighthouse Complex, Sagar Island (Station 1) is due to its location at the con¯uence of the River Hooghly and the Bay of Bengal. It is subjected to much more seawater penetration during high tide compared to Kachubaria and Haldia port complex, which are located at 30 and 50 km upper stretches of the estuary, respectively. The lowest salinity at Haldia port complex throughout the year is because it experiences minimum seawater penetration among the three stations. Barnacles are known to have tolerance to a wide range of salinity regimes (Gilles & Pequeux, 1989) and, particularly, the species B. balanoides is reported to be able
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Fig. 1. Seasonal variation of cytosolic (a) catalase, (b) superoxide dismutase (SOD) and (c) nicotinamide adenine dinucleotide hydrogen (NADH)-DT diaphorase in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
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Fig. 2. Seasonal variation of cytosolic Glutathione-S-transerase (GST) in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
to survive in the study area during the monsoon at a salinity range of 1±4 ppt (Mitra et al., 1995). The barnacle, B. balanoides, seems to possess a speci®c antioxidant enzyme system that has a conspicuous seasonal behavior. These seasonal variations might also be linked to the ¯uctuating physiological status of these animals. In barnacles, particularly in B. balanoides, proteins, lipid and carbohydrate, but not DNA, increase from late March to June (pre-monsoon period or summer), when food is most abundant in the environment (Sastry, 1989). They gradually decrease from October to late February±March (post-monsoon period or winter), presumably due to partial transfer of food from storage organs to the newly developing ovary and later on to the fertilized eggs during this period (Barnes, 1980; Sastry, 1989). The decreasing activity of antioxidant enzymes in the organisms during the postmonsoon period could depend upon physiological factors like gonad maturation and food availability. A similar seasonal variation of antioxidant enzymes occurs in mussels (bivalve mollusc) because of seasonal physiological changes (Power & Sheehan, 1996; Regoli, 1998; SoleÂ, Porte, & Albaiges, 1995b; Viarengo, Canesi, Pertica, & Livingstone, 1991). Seasonal variations in antioxidant defense systems in digestive tissue appear to have a inverse relationship to the level of microsomal lipid peroxidation. An increase in antioxidant enzyme activity coincides with a reduction in lipid peroxide formation. The reduction of the antioxidant defense systems could be responsible for an increased susceptibility to oxidative stress during the postmonsoon period, although other factors like the variations of the reduced co-enzyme
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Fig. 3. Seasonal variation of microsomal lipid peroxidation in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
Table 3 Correlation coecients (r) between physico-chemical parameters of the aquatic environment and biochemical indices in barnacle, Balanus balanoidesa Enzyme
Water temperature
Salinity
pH
Dissolved oxygen
Catalase
0.98*
0.19
0.25
0.40
SOD
0.92*
0.31
0.49
0.45
NADH-DT Diaphorase
0.98*
0.14
0.29
0.35
GST
0.65
0.43
0.66
0.07
a Data of two relatively uncontaminated sites (Stations 1 and 2) are used for the analysis. SOD, superoxide dismutase; NADH-DT, nicotinomide adenine dinucleotide hydrogen-DT; GST, glutathioneS-tranferase. *P<0.01 (n=6).
dependent oxyradical production (Livingstone et al., 1990), as well as the polyunsaturated fatty acid composition of the membranes as reported in molluscs (Gabbot, 1983), could also be involved. The actual picture of oxidative stress could be much more complex because the malondialdehyde assay only measures part of the oxidative stress, and more forms of oxidative stress could also occur.
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Table 4 Correlation coecients (r) between polyaromatic hydrocarbons concentration in soft body tissue and biochemical indices in barnacle, Balanus balanoides Catalase
Superoxide dismutase
DT-diaphorase
Glutathione-S-tranferase
0.79*
0.71*
0.34*
0.63
*P<0.05 (n=9).
Among the physico-chemical parameters, only temperature correlated signi®cantly with the behavior of antioxidant enzymes (Table 3). However, it cannot explain the dierence in the activities of antioxidant enzymes between contaminated and uncontaminated sites because of very little temperature dierence among the three stations. No signi®cant dierence in the activities of antioxidant enzymes as well as GST was observed between Stations 1 and 2 despite having a widely dierent salinity level. Thus, salinity is unlikely to be the factor causing changes in activities of antioxidant enzymes between contaminated and uncontaminated sites. The higher PAH concentration in the barnacles of Station 3 compared to Stations 1 and 2 is evidence of organic pollution in the former area, in association with industrial activity. The seasonal variation of PAH concentration is likely to be related to the seasonal variations in lipid content in the body (Meador, Stein, Reichert, & Varanasi, 1995), a pattern similar to the seasonal pattern of antioxidant enzymes and GST. From our results, it seems that the continuously elevated levels of antioxidant enzymes (particularly catalase and SOD) and GST activities, as well as microsomal lipid peroxidation (although not as pronounced as catalase and SOD) in digestive tissue of barnacles from the polluted site throughout the year, are a direct eect of environmental xenobiotics and not due to any ¯uctuation in any physico-chemical parameters. The changes in the antioxidant enzymes indicate a situation in vivo of enhanced oxyradical generation, and organic xenobiotics might be responsible for it, at least partially. Induction of antioxidant enzymes in response to contaminant-mediated reactive oxygen species production and oxidative stress has been proposed as a possible biomarker of pollution in aquatic organisms (Hugget, Kimerle, Mehrle, & Bergman, 1992: Lemaire & Livingstone, 1993). Porte et al. (1991) reported signi®cant induction of catalase (100% induction) and SOD (39% induction) in bivalves, Mytilus galloprovincialis, contaminated with PAHs compared to the relatively uncontaminated populations and suggested their potential role in environmental monitoring. Sole et al. (1995a) also reported a similar induction of catalase and SOD in Mytilus sp. in ®eld studies in Spain. In both cases, signi®cant correlation between catalase and SOD activities and tissue PAHs was observed. Therefore, our results are consistent with those from other studies, which have indicated an elevated level of antioxidant enzymes in response to PAH contamination. However, we could not detect microsomal NADPH-DT diaphorase in barnacle, B. balanoides, which is known to play an important protective role against organic pollution in bivalve molluscs. There is hardly any information available about the role of cytosolic NADH-DT diaphorase in marine invertebrates and the very small but consistent induction in cytosolic NADH-DT
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Fig. 4. Regression of cytosolic (a) catalase and (b) superoxide dismutase (SOD) in digestive tissue on polyaromatic hydrocarbon (PAH) concentration in whole body tissue in Balanus balanoides. The regression equations (S.E. of estimate of y on x in square parenthesis) are (a) y=0.58+2.12 [0.85] and (b) y=0.61+3.78 [1.15].
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diaphorase in barnacles of the polluted site compared to the control site was found to be statistically insigni®cant. The biotransformation enzyme, GST, showed an inconsistent pattern of signi®cant induction in B. balanoides. Despite some evidence for a biomarker role for GST activity in bivalves (Sheehan et al., 1994), this result is consistent with other studies in re¯ecting the lack of persistent elevated GST activity in bivalves exposed to PAHs (Michel, Suteau, Robertson, & Narbonne, 1993). In summary, given the magnitude of the changes of catalase and SOD observed in our study, it can be suggested that these two antioxidant enzymes could be potential biomarkers for the evaluation of contaminated marine ecosystems. The results also indicate that like bivalve molluscs and gastropods, the barnacles could also be a potential bioindicator of organic pollution, although further research is needed, particularly in the area of its response to speci®c xenobiotics.
Acknowledgements The authors gratefully acknowledge the Ministry of Environment & Forests, Government of India, for sponsoring the work.
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Livingstone, D. R., Garcia Martinez, P., Michel, X., Narbonne, J. F., O'Hara, S., Ribera, D., & Winston, G. W. (1990). Oxyradical production as pollution-mediated mechanism of toxicity in the common mussel Mytilus edulis L. and other molluscs. Functional Ecology, 4, 415±424. Meador, J. P., Stein, J. E., Reichert, W. L., & Varanasi, U. (1995). Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Reviews of Environmental Contamination and Toxicology, 143, 79±165. Michel, X., Suteau, P., Robertson, L. W., & Narbonne, J. F. (1993). Eects of benzo(a)pyrene, 3,30 ,4,40 tetrachlorobiphenyl and 2,20 ,4,40 ,5,50 -hexachlorobiphenyl on the xenobiotic metabolizing enzymes in the mussel (Mytilus galloprovincialis). Aquatic Toxicology, 27, 335±344. Mitra, A., & Choudhury, A. (1993). Trace metals in macrobenthic molluscs of the Hooghly Estuary, India. Marine Pollution Bulletin, 26(9), 521±522. Mitra, A., Trivedi, S., Chaudhuri, A., Bag, M., Ghosh, I., & Choudhury, A. (1995). Balanus balanoides as an indicator of heavy metals. Indian Journal of Environmental Health, 37(1), 42±45. Paoletti, F., & Mocali, A. (1990). Determination of superoxide dismutase activity by purely chemical system based on NAD(P)H oxidation. In L. Packer & A. N. Glazer, Methods in enzymology, (Vol. 186, pp. 209±221). New York: Academic Press. Porte, C., Sole, M., Albaiges, J., & Livingstone, D. R. (1991). Responses of mixed-function oxygenase and antioxidase enzyme system of Mytilus sp. to organic pollution. Comparative Biochemistry and Physiology, 100C(1/2), 183±186. Power, A., & Sheehan, D. (1986). Seasonal variation in the antioxidant defense systems of gill and digestive gland of the blue mussel, Mytilus edulis. Comparative Biochemistry and Physiology, 114C, 99±103. Rodriguez-Ariza, A., Martinez-Lara, E., Pascual, P., Pedrajas, J. R., Abril, N., Dorado, B. G., Toribio, F., Barcena, J. A., Peinado, J., Pueyo, C., & Lopez-Barea, J. (1993). Biochemical and genetic indices of marine pollution in Spanish littoral. Proceedings of the Second European Conference on Ecotoxicology (pp. 109±116). Amsterdam, The Netherlands. Regoli, F. (1998). Trace metals and antioxidant enzymes in gills and digestive gland of the Mediterranean mussel, Mytilus galloprovincialis. Archives of Environmental Contamination and Toxicology, 34, 48±63. Regoli, F., Nigro, M., Bertoli, E., Principaro, G., & Orlando, E. (1997). Defenses against oxidative stress in the Antarctic scallop, Adamussium colbecki and eects of acute exposure to metals. Hydrobiologia, 355(1±3), 139±144. Sastry, A. N. (1989). Ecological aspects of reproduction. In J. F. Vernberg & W. B. Vernberg, Biology of Crustacea (Vol. 8, pp. 179±269). New York: Academic Press. Sato, R., & Omura, T. (1978). Cytochrome P-450 (pp. 230±246). New York: Academic Press. Sheehan, D., McIntosh, J., Power, A., & Fitzpatrick, P. J. (1994). Drug metabolizing enzymes of mussels as bioindicators of chemical pollution. Biochemical Society Transactions, 23, 419±422. SoleÂ, M., Porte, C., & Albaiges, J. (1994). Mixed function oxygenase components and antioxidant enzymes in dierent marine bivalves: its relation with contaminant body burdens. Aquatic Toxicology, 30, 271±283. SoleÂ, M., Porte, C., & Albaiges, J. (1995a). The use of biomarkers for assessing the eects of organic pollution in mussels. Science of the Total Environment, 159, 147±153. SoleÂ, M., Porte, C., & Albaiges, J. (1995b). Seasonal variation in the mixed function oxygenase system and antioxidant enzymes of the mussel, Mytilus galloprovincialis. Environmental Toxicology and Chemistry, 14, 157±164. Stegeman, J. J., & Kaplan, H. B. (1981). Mixed function oxygenase activity and benzo(a)pyrene metabolism in the barnacle, Balanus eburneus (Crustacea: Cirripedia). Comparative Biochemistry and Physiology, 68C, 55±61. Strickland, J. D. H., & Parsons, T. R. (1968). A practical handbook of seawater analysis. Ottawa: Fisheries Research Board of Canada. Viarengo, A., Canesi, L., Pertica, M., & Livingstone, D. R. (1991). Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels. Comparative Biochemistry and Physiology, 100C(1/2), 187±190.
Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons S. Niyogi a, S. Biswas a, S. Sarker b, A.G. Datta a,* a Department of Life Science & Biotechnology, Jadavpur University, Calcutta 700032, India Indian Institute of Chemical Biology, 4 Raja S.C. Mallick Road, Jadavpur, Calcutta 700032, India
b
Received 25 October 1999; received in revised form 19 March 2000; accepted 19 July 2000
Abstract Seasonal variations in the antioxidant enzymes (catalase, superoxide dismutase [SOD], NADH-DT diaphorase), biotransformation enzyme, glutathione-S-transferase (GST) and microsomal lipid peroxidation in digestive tissue of barnacle, Balanus balanoides, from polluted and non-polluted populations have been evaluated. Relationships with accumulated polyaromatic hydrocarbon (PAH) concentration in barnacle tissues and environmental parameters (water temperature, salinity, dissolved oxygen concentration, water pH) were determined. As a general trend, maximum antioxidant enzyme and GST activities were detected in the pre-monsoon period or summer (March±June) followed by a gradual decrease during the monsoon (July±October) with a minimum in the post-monsoon period or winter (November± February). This pattern was similar to tissue concentrations of PAHs, resulting in a signi®cant positive correlation with antioxidant enzymes, mainly catalase and SOD. Microsomal lipid peroxidation exhibited an almost reverse trend of seasonal variation to that of antioxidant enzyme activities indicating an enhanced susceptibility of barnacle tissues to oxidative stress. Among the environmental parameters, only water temperature seemed to have a signi®cant eect on observed variations of antioxidant enzymes and GST activities. The barnacles from polluted and non-polluted populations exhibited seasonal dierences in the activities of all the enzymes studied, particularly catalase, SOD and GST, suggesting the possibility of some biochemical adaptation in organisms from a chronically polluted environment. The results indicated that antioxidant defense components, catalase and SOD, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The * Corresponding author. Tel.: +91-33-474-9696. E-mail address: [email protected] (A.G. Datta). 0141-1136/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00257-9
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results also suggested the potentiality of barnacle, B. balanoides, as a bioindicator organism against organic pollution. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Hooghly Estuary; Barnacle; Seasonal variation; Physico-chemical parameters; PAHs; Antioxidant enzymes; GST; Biomarkers
1. Introduction Pollution in the marine environment aects not only aquatic life as such but also poses a serious threat to all life on the planet. The rapid advancement of industrial development worldwide has led to a continual in¯ux of xenobiotics into oceans, particularly polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc., which may ultimately upset the fragile balance of life in the seas. Estuaries and coastal waters are particularly at risk from such anthropogenic pollution. It is always very dicult from only contaminant body burden data to obtain information about their signi®cance upon animal health. Therefore, techniques for measuring biological eects are critical for any pollution monitoring program. Among various biochemical systems, monitoring the cytochrome P450 monooxygenase or mixed-function oxygenase (MFO) system is one of the most widely accepted methods for detecting organic pollution in marine environment. The MFO system is an apparently universally distributed enzyme system involved in the detoxi®cation as well as in some cases, activation of xenobiotics such as PAHs (Sato & Omura, 1978). Recently, it has been suggested that antioxidant as well as phase II enzymes like glutathione-S-transferase (GST) in conjunction with MFO system can also be studied to build up a more comprehensive picture of toxic eect and response (Foerlin, Lemaire, Livingstone, Forlin, & Andersson, 1995; Livingstone, 1991; Porte, Sole, Albaiges, & Livingstone, 1991; Regoli, Nigro, Bertoli, Principaro, & Orlando, 1997; Rodriguez-Ariza et al., 1993; Sheehan, McIntosh, Power, & Fitzpatrick, 1994; SoleÂ, Porte, & Albaiges, 1994, 1995a). In this aspect, a majority of the published works are concerned with bivalve molluscs, particularly mussels and oysters, but there is very little information available about these systems in other marine invertebrates like lower order crustaceans, a large, diverse, and complex group of animals. Barnacles (sub class: Cirripedia; Order: Thoracica) (Barnes, 1980) comprise a unique and important group in the class Crustacea. Exclusively marine and having worldwide distribution and great abundance, they are the only group of exclusively sessile, hermaphroditic crustaceans which make them suitable as bioindicator organisms. Although Mitra, Trivedi, Chaudhuri, Bag, Ghosh, and Choudhury (1995) suggested the usefulness of barnacles as an indicator of heavy metal pollution, there is no information available about the potentiality of these organisms for the biomonitoring of organic pollution. Stegeman and Kaplan (1981) characterized cytochrome P450 and its components in acorn barnacle, Balanus eburneus, but the status of antioxidant and other biotransformation enzymes in these organisms and their response to organic pollution is virtually unknown. In this paper, we report the seasonal variations of the activities of three cytosolic antioxidant enzymes [Catalase
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(EC 1.11.1.6), superoxide dismutase SOD EC 1.15.1.1) and DT-diaphorase (EC 1.6.99.2)], a cytosolic phase II enzyme, GST (EC 2.5.1.18) and microsomal lipid peroxidation in the digestive tissue (digestive gland along with intestine) of barnacle, Balanus balanoides, collected from two dierent locations along Hooghly Estuary, India. Seasonal variations of physico-chemical parameters of ambient water (surface water temperature, salinity, pH, dissolved oxygen) and the PAH concentrations in whole-body tissues of barnacles from the two locations were also determined. 2. Site characteristics The Hooghly Estuary (88 000 ±89 280 E longitude and 21 000 ±22 300 N latitude), which supports the world's largest and most magni®cent mangrove block, the Sunderbans, is a typical and very conspicuous ecosystem of the Indian subcontinent. This unique estuary and the Sunderbans are among the world's most precious genetically diverse ecosystems. With the rapid emergence of the Haldia port complex as a major oil disembarkment terminal in eastern India and the industrialization of the surrounding areas, including development of India's largest oil re®nery, the estuary has become vulnerable to pollution, particularly due to petroleum-derived hydrocarbons including PAHs. This Haldia port complex area was chosen as the polluted site of our study (Station 3). The Old Lighthouse Complex at Sagar Island, a remote place inside the mangrove block at the lower stretch of Hooghly Estuary, which is more than 50 km away from the Haldia port complex, was chosen as the reference site (Station 1). Kachubaria, another remote area situated at the northern tip of Sagar Island was also considered for this study (Station 2). Both these areas are less polluted than the Haldia port complex due to the absence of any nearby industrial activity. 3. Methods and materials Barnacles, B. balanoides, were collected from all the three stations (period of study: November 1997±October 1998). Samples of consistent size range (diameter of the base of shell Ð 1.4±1.5 cm) were taken every time as recommended by the guidelines of the Joint Monitoring Group of the Oslo and Paris conventions (Mitra & Choudhury, 1993). Sampling was done twice a month from all the three stations during low tide. The samples were transferred to the laboratory in living condition and processed immediately. Animals were removed from calcareous shells, the digestive tissues (digestive gland with intestine) were dissected out, and ¯uids were rinsed from the intestine. Dissected digestive tissues of 2 g (pooled) were immediately homogenized in 5 vol. of 65 mM Na-phosphate buer, 0.15 M KCl, 3 mM MgCl2, 0.1 mM phenyl methyl sulfonyl ¯uoride, pH 7.0 using a PotterElvehjam-type glass te¯on homogeniser and centrifuged at 12,000g for 30 min at 4 C. The 12,000g supernatant was again centrifuged at 100,000g for 90 min at 4 C. The 100,000g supernatants or cytosol was subsequently used for the
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evaluation of enzyme activities. Catalase activity was assayed by the method of Greenwald (1985). The reaction mixture contained 50 mM phosphate buer (pH 7.0) and 50 mM H2O2. The reaction rate was measured at 240 nm. One unit of catalase activity was de®ned as 1 mmol of H2O2 degraded min 1 g 1 wet weight. SOD activity was determined by the method of Paoletti and Mocali (1990). The inhibitory eect of SOD on the superoxide generation in presence of ethylene-diamine tetra acetic acid (EDTA)±manganese (II) chloride (2.5/1.25 mM), mercaptoethanol (1 mM) and reduced nitcoinamide adenine dinucleotidephosphate (NADPH) (0.3 mM) in triethanolamine-diethanolamine (100 mM each) hydrochloric acid± (HCl) buer (pH 7.4) was analyzed spectrophotometrically at 340 nm. The assays were run without and with dierent volumes of enzyme sample to determine 50% inhibition. One unit of SOD activity was calculated as the amount of enzyme required to inhibit the rate of NADPH oxidation of the control by 50%. Cytosolic nicotinamide adenine dinucleotide hydrogen (NADH-TD) diaphorase activity was measured as dicumarol inhibitable NADH±dichlorophenolindophenol (DCPIP) reductase activity (Livingstone et al., 1990). The reaction mixture contained 50 mM Tris±HCl (pH 7.6), 0.3 mM NADH and 40 mM DCPIP. The decrease in absorbance was monitored at 600 nm with and without 100 mM dicumarol in 0.15% NaOH to determine the actual rate of activity of the enzyme. One unit of NADH-DT diaphorase activity was de®ned as nanomols of DCPIP reduced per minute per milligram protein. Microsomal NADPH-DT diaphorase was measured similarly replacing NADH with NADPH. GST was measured according to Habig, Pabst and Jakoby (1974) using 1-chloro-2-4-dinitrobenzene (CDNB) as substrate. The standard assay mixture contained 0.1 M phosphate buer (pH 6.5), 1 mM reduced glutathione (GSH), 1 mM EDTA and 1 mM CDNB. The complete assay mixture without enzyme was used as a control. The rate of reaction was measured as the increase in absorbance at 340 nm. The chemical rate of reaction, determined in the absence of sample, was subtracted from the sample. One unit of the enzyme was de®ned as 1 nmol of GSH conjugated min 1 mg 1 protein. The 100,000g pellets or microsomes were washed three to four times in 0.1 M phosphate buer, pH 7.3 and ®nally resuspended in 2 ml of the same buer. Lipid peroxidation in microsomal fraction was measured as malondialdehyde equivalent using trichloroacetic acid (TCA), thiobarbituric acid (TBA) and (HCl) reagent (TBA±TCA reagent: 0.375% w/v TBA, 15% w/v TCA and 0.25 N HCl) (Buege & Aust, 1978). Microsomal suspension (1 ml) was mixed with 2 ml of TBA±TCA reagent and heated in a boiling waterbath for 15 min. After cooling, the ¯occulant precipitate was removed by centrifugation at 1000g for 10 min. Finally the malondialdehyde concentration in the supernatant fraction was determined spectrophotometrically at 535 nm using an extinction coecient of 1.56105 M 1 cm 1. The results are expressed as nanomols MDA g 1 wet weight. Proteins in all samples were determined by the method of Bradford (1976) with bovine serum albumin as standard. Chemical analyses of barnacle tissues were carried out, basically according to the method of Porte et al. (1991) with slight modi®cations. The whole soft body tissues of barnacles (1 g) were homogenized and Soxhlet-extracted for 12 h with n-hexane and methylene chloride (4:1). The extract was treated with Na2SO4 overnight to
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remove remaining water and ®nally applied to a silica gel column to purify it. PAH concentration was determined in the corresponding fraction by UV ¯uorescence, emission and excitation intensities being measured at 360 and 310 nm, respectively, and expressed as mg g 1 wet weight (chrysene equivalents). Internal standard (chrysene, 95% pure, procured from Sigma Chemicals Ltd., St Louis, Missouri, USA) was added to tissue homogenate before extraction as a quality control method and the percentage recovery was 88.5 (1.8 S.E.M., n=8). All physico-chemical parameters were assessed during each sampling. Surface water temperature was measured with a Celsius thermometer (ranging 0±100 C). The surface water salinity around the sampling stations were estimated by employing the ``Mohr±Knudssen method'' (Strickland & Parsons, 1968). The correction factor was determined by titrating silver nitrate solution against standard seawater [IAPO standard seawater service, Charlottenlund, Stot, Denmark (salinity 19.376)]. The pH of the ambient media of both stations was determined on the spot by a portable pH meter (sensitivity=0.02 units). Dissolved oxygen in the ambient water was determined by the method as described in Strickland and Parsons. Statistical signi®cance was determined by Student's `t test'. 4. Results Among the physico-chemical parameters (Table 1), salinity exhibited a distinct seasonal pattern in all the three stations. Maximum values were recorded during the pre-monsoon period (March±June), followed by minimum values during the monsoon period (July±October) and again a slight increase during post-monsoon (November±February). Salinity was higher at Station 1 throughout the year followed by Stations 2 and 3, respectively. Other physico-chemical parameters (surface water temperature, pH, dissolved oxygen) exhibited little dierence between the three stations. Surface water temperature was similar during the pre-monsoon and monsoon periods with a moderate decrease during the post-monsoon period. Aquatic pH and the dissolved oxygen decreased slightly during the monsoon period, but otherwise maintained a consistent pattern throughout the year. The PAH concentration in the soft body tissues B. balanoides (Table 2) exhibited high values (six to nine times higher) in animals collected from Station 3 compared to Stations 1 and 2 throughout the year. The PAH concentration in soft body tissues also showed a distinct seasonal pattern with a maxima during the pre-monsoon period followed by gradual decrease during the monsoon to post-monsoon period. All the antioxidant enzymes in the digestive tissues of barnacles at all three stations exhibited a similar seasonal pro®le throughout the year (Fig. 1a, b, c). Maximum activity occurred during the pre-monsoon period with a gradual decrease during monsoon to post-monsoon. The seasonal variation of the biotransformation enzymes, GST, was not as distinct as the antioxidant enzymes (Fig. 2). Although the maxima was observed during the pre-monsoon period, there was very little variation in GST activities between monsoon and post-monsoon periods. Microsomal lipid peroxidation in digestive tissues of barnacles showed a reverse pro®le to that of antioxidant
18
Season
Water Temperature ( C)
Salinity (ppt)
Station 1
Station 1
Station 2
Station 3
Station 2
Dissolved oxygen (mg l 1)
pH Station 3
Station 1
Station 2
Station 3
Station 1
Station 2
Station 3
Pre-monsoon 30.70.84 32.20.74 31.10.69 26.90.36 15.20.49 8.340.68 8.280.04 8.200.04 8.190.03 5.890.26 5.410.27 5.620.20 Monsoon 29.90.43 29.60.65 30.10.66 18.11.25 5.40.42 1.50.76 7.970.06 7.980.03 7.880.05 5.280.22 6.020.31 5.150.10 Post-monsoon 24.80.82 25.10.47 24.30.52 22.40.53 9.60.81 2.90.43 8.210.03 8.010.04 8.080.04 5.530.15 5.080.30 5.210.13 a
Values are expressed as meansS.E.M. (n=8).
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Table 1 Seasonal variation of physico-chemical parameters of surface water in three stationsa
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Table 2 Seasonal variation of polyaromatic hydrocarbon concentration in soft body tissue of barnacle, Balanus balanoidesa Season
Station 1
Station 2
Station 3
Pre-monsoon Monsoon Post-monsoon
0.70.08 0.50.07 0.40.06
0.80.10 0.60.06 0.50.06
5.10.71 4.50.87 3.10.22
a
Values are expressed as mg g
1
wet weight (chrysene equivalents) and meansS.E.M. (n=8).
enzymes (Fig. 3). It was lowest during the pre-monsoon period with subsequent increase from the monsoon to post-monsoon period. Our study also revealed that the antioxidant enzymes and GST showed persistent induction in barnacles at the highly contaminated Station 3 compared to Stations 1 and 2. Catalase and SOD maintained signi®cant induction at Station 3 compared to Station 1 (control station) throughout the year. Cytosolic NADH-DT diaphorase exhibited less and insignificant induction. Microsomal NADPH-DT diaphorase could not be detected in barnacles. GST induction was signi®cantly dierent at Station 3 compared to Station 1 during the pre-monsoon period only. Microsomal lipid peroxidation also maintained a consistent elevated level at Station 3 compared to the control station although the dierence was signi®cant only in the post-monsoon period. However, no signi®cant dierence between the activities of any of these enzymes and microsomal lipid peroxidation was observed in barnacles collected from Station 1 and 2. Among the physico-chemical parameters, only surface water temperature showed signi®cant correlation with biochemical parameters (Table 3) whereas PAH concentration in soft body tissue of barnacles correlated signi®cantly with catalase and SOD (Table 4). The linear regression analysis of catalase and SOD on tissue PAH concentration exhibited a positive slope in both cases (Fig. 4a, b). 5. Discussion The results indicate that the seasonal monsoonal cycle in the Indian subcontinent seems to have some eect on physico-chemical parameters of the aquatic environment, particularly on salinity and to some extent on pH. The decrease in salinity and pH in the monsoon period is due to massive rainfall that causes a considerable increase in fresh water run o from the upper stretch of the estuary. The higher salinity at Old Lighthouse Complex, Sagar Island (Station 1) is due to its location at the con¯uence of the River Hooghly and the Bay of Bengal. It is subjected to much more seawater penetration during high tide compared to Kachubaria and Haldia port complex, which are located at 30 and 50 km upper stretches of the estuary, respectively. The lowest salinity at Haldia port complex throughout the year is because it experiences minimum seawater penetration among the three stations. Barnacles are known to have tolerance to a wide range of salinity regimes (Gilles & Pequeux, 1989) and, particularly, the species B. balanoides is reported to be able
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Fig. 1. Seasonal variation of cytosolic (a) catalase, (b) superoxide dismutase (SOD) and (c) nicotinamide adenine dinucleotide hydrogen (NADH)-DT diaphorase in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
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Fig. 2. Seasonal variation of cytosolic Glutathione-S-transerase (GST) in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
to survive in the study area during the monsoon at a salinity range of 1±4 ppt (Mitra et al., 1995). The barnacle, B. balanoides, seems to possess a speci®c antioxidant enzyme system that has a conspicuous seasonal behavior. These seasonal variations might also be linked to the ¯uctuating physiological status of these animals. In barnacles, particularly in B. balanoides, proteins, lipid and carbohydrate, but not DNA, increase from late March to June (pre-monsoon period or summer), when food is most abundant in the environment (Sastry, 1989). They gradually decrease from October to late February±March (post-monsoon period or winter), presumably due to partial transfer of food from storage organs to the newly developing ovary and later on to the fertilized eggs during this period (Barnes, 1980; Sastry, 1989). The decreasing activity of antioxidant enzymes in the organisms during the postmonsoon period could depend upon physiological factors like gonad maturation and food availability. A similar seasonal variation of antioxidant enzymes occurs in mussels (bivalve mollusc) because of seasonal physiological changes (Power & Sheehan, 1996; Regoli, 1998; SoleÂ, Porte, & Albaiges, 1995b; Viarengo, Canesi, Pertica, & Livingstone, 1991). Seasonal variations in antioxidant defense systems in digestive tissue appear to have a inverse relationship to the level of microsomal lipid peroxidation. An increase in antioxidant enzyme activity coincides with a reduction in lipid peroxide formation. The reduction of the antioxidant defense systems could be responsible for an increased susceptibility to oxidative stress during the postmonsoon period, although other factors like the variations of the reduced co-enzyme
22
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Fig. 3. Seasonal variation of microsomal lipid peroxidation in digestive tissue of Balanus balanoides from three stations along the Hooghly Estuary. Signi®cant dierences in comparison with Station 1 indicated by an asterisk, P<0.05.
Table 3 Correlation coecients (r) between physico-chemical parameters of the aquatic environment and biochemical indices in barnacle, Balanus balanoidesa Enzyme
Water temperature
Salinity
pH
Dissolved oxygen
Catalase
0.98*
0.19
0.25
0.40
SOD
0.92*
0.31
0.49
0.45
NADH-DT Diaphorase
0.98*
0.14
0.29
0.35
GST
0.65
0.43
0.66
0.07
a Data of two relatively uncontaminated sites (Stations 1 and 2) are used for the analysis. SOD, superoxide dismutase; NADH-DT, nicotinomide adenine dinucleotide hydrogen-DT; GST, glutathioneS-tranferase. *P<0.01 (n=6).
dependent oxyradical production (Livingstone et al., 1990), as well as the polyunsaturated fatty acid composition of the membranes as reported in molluscs (Gabbot, 1983), could also be involved. The actual picture of oxidative stress could be much more complex because the malondialdehyde assay only measures part of the oxidative stress, and more forms of oxidative stress could also occur.
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Table 4 Correlation coecients (r) between polyaromatic hydrocarbons concentration in soft body tissue and biochemical indices in barnacle, Balanus balanoides Catalase
Superoxide dismutase
DT-diaphorase
Glutathione-S-tranferase
0.79*
0.71*
0.34*
0.63
*P<0.05 (n=9).
Among the physico-chemical parameters, only temperature correlated signi®cantly with the behavior of antioxidant enzymes (Table 3). However, it cannot explain the dierence in the activities of antioxidant enzymes between contaminated and uncontaminated sites because of very little temperature dierence among the three stations. No signi®cant dierence in the activities of antioxidant enzymes as well as GST was observed between Stations 1 and 2 despite having a widely dierent salinity level. Thus, salinity is unlikely to be the factor causing changes in activities of antioxidant enzymes between contaminated and uncontaminated sites. The higher PAH concentration in the barnacles of Station 3 compared to Stations 1 and 2 is evidence of organic pollution in the former area, in association with industrial activity. The seasonal variation of PAH concentration is likely to be related to the seasonal variations in lipid content in the body (Meador, Stein, Reichert, & Varanasi, 1995), a pattern similar to the seasonal pattern of antioxidant enzymes and GST. From our results, it seems that the continuously elevated levels of antioxidant enzymes (particularly catalase and SOD) and GST activities, as well as microsomal lipid peroxidation (although not as pronounced as catalase and SOD) in digestive tissue of barnacles from the polluted site throughout the year, are a direct eect of environmental xenobiotics and not due to any ¯uctuation in any physico-chemical parameters. The changes in the antioxidant enzymes indicate a situation in vivo of enhanced oxyradical generation, and organic xenobiotics might be responsible for it, at least partially. Induction of antioxidant enzymes in response to contaminant-mediated reactive oxygen species production and oxidative stress has been proposed as a possible biomarker of pollution in aquatic organisms (Hugget, Kimerle, Mehrle, & Bergman, 1992: Lemaire & Livingstone, 1993). Porte et al. (1991) reported signi®cant induction of catalase (100% induction) and SOD (39% induction) in bivalves, Mytilus galloprovincialis, contaminated with PAHs compared to the relatively uncontaminated populations and suggested their potential role in environmental monitoring. Sole et al. (1995a) also reported a similar induction of catalase and SOD in Mytilus sp. in ®eld studies in Spain. In both cases, signi®cant correlation between catalase and SOD activities and tissue PAHs was observed. Therefore, our results are consistent with those from other studies, which have indicated an elevated level of antioxidant enzymes in response to PAH contamination. However, we could not detect microsomal NADPH-DT diaphorase in barnacle, B. balanoides, which is known to play an important protective role against organic pollution in bivalve molluscs. There is hardly any information available about the role of cytosolic NADH-DT diaphorase in marine invertebrates and the very small but consistent induction in cytosolic NADH-DT
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Fig. 4. Regression of cytosolic (a) catalase and (b) superoxide dismutase (SOD) in digestive tissue on polyaromatic hydrocarbon (PAH) concentration in whole body tissue in Balanus balanoides. The regression equations (S.E. of estimate of y on x in square parenthesis) are (a) y=0.58+2.12 [0.85] and (b) y=0.61+3.78 [1.15].
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diaphorase in barnacles of the polluted site compared to the control site was found to be statistically insigni®cant. The biotransformation enzyme, GST, showed an inconsistent pattern of signi®cant induction in B. balanoides. Despite some evidence for a biomarker role for GST activity in bivalves (Sheehan et al., 1994), this result is consistent with other studies in re¯ecting the lack of persistent elevated GST activity in bivalves exposed to PAHs (Michel, Suteau, Robertson, & Narbonne, 1993). In summary, given the magnitude of the changes of catalase and SOD observed in our study, it can be suggested that these two antioxidant enzymes could be potential biomarkers for the evaluation of contaminated marine ecosystems. The results also indicate that like bivalve molluscs and gastropods, the barnacles could also be a potential bioindicator of organic pollution, although further research is needed, particularly in the area of its response to speci®c xenobiotics.
Acknowledgements The authors gratefully acknowledge the Ministry of Environment & Forests, Government of India, for sponsoring the work.
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