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Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura)
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Ecotoxicology and Environmental Safety 65 (2006) 201–208 www.elsevier.com/locate/ecoenv
Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura) L.J. Dewesa,b,, J.Z. Sandrinic,d, J.M. Monserratb,c,d, J.S. Yunesa,b Unidade de Pesquisa em Cianobacte´rias, Fundac- a˜o Universidade Federal do Rio Grande (FURG), Av. Ita´lia Km 8, Campus Carreiros, Hidroquı´mica, Caixa Postal 474, Rio Grande, RS, Brazil b Programa de Po´s-Graduac- a˜o em Oceanografia Biolo´gica, FURG, Brazil c Programa de Po´s-Graduac- a˜o em Cieˆncias Fisiolo´gicas, Fisiologia Animal Comparada, FURG, Brazil d Departamento de Cieˆncias Fisiolo´gicas, FURG, Brazil
a
Received 2 December 2004; received in revised form 8 July 2005; accepted 11 July 2005 Available online 31 August 2005
Abstract Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 mg kg1) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 mg kg1 of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 mg kg1. LPO levels increased in doses equal to or higher than 34 mg kg1 after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 mg kg1), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 mg kg1. After 12 h of exposure to 860 mg kg1 of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.1770.56 mg kg1. In crabs exposed to doses higher than 172 mg kg1 during 72 h this value raised to 32.1474.12 mg kg1. The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances. r 2005 Elsevier Inc. All rights reserved. Keywords: Microcystins; Chasmagnathus granulatus; Hepatopancreas; Glutathione S-transferases; Lipid peroxidation; Glycogen
1. Introduction The Patos Lagoon in southern Brazil is the second largest freshwater body in South America. Its waters are Corresponding author. Fax: +55 53 2336737.
E-mail address: [email protected] (L.J. Dewes). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.07.013
used for drinking, leisure, navigation, and agriculture by more than a million inhabitants. Furthermore, the lagoon estuarine region is a very rich and complex ecosystem with several fish, mollusk, and crustacea species being commercially exploited (Yunes et al., 2004). Toxic blooms of cyanobacteria, usually dominated by Microcystis aeruginosa, have been registered in
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the Patos Lagoon (RS, Brazil) in the past two decades, and the microcystins released in the water have reached concentrations as high as 289 mg L1 (Minillo et al., 2000). Microcystins are known to inhibit phosphatases of types 1 and 2A, resulting in excessive phosphorylation of cytoskeletal filaments, leading to liver failure (Carmichael, 1992). These toxins are cyclic heptapeptides, with a generic structure of cyclo-(D-Ala1-X2-DMeAsp3-Y4-Adda5-D-Glu6-Mdha7), where X and Y are variable L-aminoacids (Sivonen and Jones, 1999). [D-Leu1]Microcystin-LR was identified as the most abundant microcystin released from the strain of M. aeruginosa used in this work (RST 9501) (Matthiensen et al., 2000). The hepatic damage caused by microcystins can be measured by some clinical enzyme markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). ALT is a transaminase that has been used as an important clinical indicator of hepatocellular injury (Solter et al., 2000). Recent evidence has shown that microcystins also induce oxidative stress and disruption of osmotic and ion regulation in crustacean species (Pinho et al., 2003; Vinagre et al., 2003). In vertebrates, microcystins are taken up into the hepatocytes by multispecific bile acid transporters (Eriksson et al., 1990; Runnegar et al., 1995), augmenting the production of reactive oxygen species (ROS), lipid peroxides, DNA damage, and activity of antioxidant enzymes (Ding et al., 1998, 2001; Gehringer et al., 2004a; Malbrouck et al., 2003; Zˇegura et al., 2003). The detoxification of microcystins in the liver is known to occur via conjugation to glutathione (GSH) by glutathione-S-transferase (GST) activity (Pflugmacher et al., 1998). It is important to note that GSH is considered one of the main nonenzymatic antioxidant and constitutes the first line of protection against ROS (Griffith, 1999; Sies, 1999). In this way, the conjugation of microcystin with GSH should represent a leak of this tripeptide from the intracellular pool, promoting the generation of oxidative stress. Microcystins still can promote oxidative stress through an increase of oxygen consumption, previously observed in aquatic organisms exposed to these toxins (Montagnolli et al., 2004), which in turn could increase ROS production. Augmented oxygen consumption can be related to a higher activity of the glycolitic pathway, through the stimulation of the enzyme glycogen phosphorylase via inhibition of protein phosphatases by microcystins. An increase of the glycolitic pathway can also promote ROS generation (Dro¨ge, 2002; Folmer et al., 2002). Up to date, little information exists about the internal concentration of microcystins in aquatic invertebrate organism after exposure to these kind of toxins and the specific effects on ALT, AST, and LDH activities. Taking into account this fact, the present study aimed to
evaluate the dose effects of microcystins in some biochemical and physiological parameters in the hepatopancreas of C. granulatus, including some related to oxidative stress as lipid peroxides levels.
2. Material and methods Adult male crabs were collected in salt marshes round the city of Rio Grande (RS, Southern Brazil, 301200 S–321100 S) in winter and spring. They were immediately transferred to the Department of Physiological Sciences (Fundac- a˜o Universidade Federal do Rio Grande, Brazil) and maintained in tanks with water at controlled temperature (20 1C) and salinity (2%), under constant aeration, for at least 1 month. Photoperiod was fixed at 12L:12D. Crabs were fed 3 days a week with ground beef. The water salinity was fixed to 2%, as previous evidence indicates that blooms of M. aeruginosa in Patos Lagoon occur in low salinities (Yunes et al., 1998). Cells of M. aeruginosa (strain RST 9501; Matthiensen et al., 2000) were cultured in BG11 medium (plus 8.82 mM of NaNO3) at 2571 1C (Rippka et al., 1979) and employed as source of microcystins. Cells were sonicated for 3 min in 100 Hz and centrifuged (10,000g, 10 min) at room temperature. The supernatant was collected and stored at 80 1C until use. Microcystins content of the extracts was determined using a commercial enzyme-linked immunoassay (ELISA) with polyclonal antibodies (EnviroLogix Inc., Portland, ME). After acclimation, crabs were employed in two different bioassays. In bioassay 1 (crabs collected in spring, mean weight 9.0672.76 g; n ¼ 69), organisms were exposed during 6 and 12 h to 860 mg kg1 of microcystins. In bioassay 2 (crabs collected in winter, mean weight 8.0272.44 g; n ¼ 69), crabs were exposed during 72 h to 34, 172, and 860 mg kg1 of microcystins. The different doses were obtained by diluting the stock solution of microcystins (obtained as described above) in crustacean saline solution (Vinagre et al., 2003). The microcystins exposed groups received 100 ml of the cyanobacterial extract by gavage, while the control groups were exposed only to crustacean saline solution. At the end of the bioassays, crabs were sacrificed and their hepatopancreas dissected immediately. Oxygen consumption was measured in samples of about 50 mg of tissue. Other pieces of hepatopancreas were stored at 80 1C for biochemical analysis. Crabs were not fed during the exposure period. Oxygen consumption was determined according to Nithart et al. (1999) through the determination of oxygen concentration in time 0 and 30 min postincubation of tissue at 20 1C in physiological solution
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plus 1 mM of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Sigma). For analysis of enzyme activity (GST, ALT, AST, and LDH), microcystins, and protein content, the hepatopancreas was homogenized (20% w/v) in a buffer containing Tris base (20 mM), EDTA (1 mM), dithiothreitol (DTT 1 mM, Sigma), sucrose (0.5 mM), and KCl (150 mM), the pH being adjusted to 7.60. Samples were centrifuged at 9000g (4 1C) for 30 min and the supernatants employed as enzyme source. For microcystin analysis, after homogenization samples were sonicated (3 min, 100 Hz), centrifuged (10 min, 10,000g), and diluted with an equal volume of chloroform. The supernatants were collected and the microcystin content determined by immunoassay. Glutathione-S-transferase (GST) activity was measured according to Habig et al. (1974) and Habig and Jakoby (1981) by evaluating the conjugation of reduced glutathione (GSH; 1 mM; Sigma) with 1-chloro-2,4dinitro benzene (CDNB, 1 mM; Sigma) at 340 nm for 1 min. Aliquots of hepatopancreas homogenates were incubated (25 1C) in phosphate buffer 0.1 M at pH 7.00. An extinction coefficient of 9.6 mM1 cm1 was employed in order to calculate the moles of CDNB conjugated. Enzyme activity was calculated considering the total protein content in the homogenate. One GST unit was defined as the amount of enzyme necessary to conjugate 1 mmol of 1-chloro-2,4-dinitrobenzene (Sigma) per minute and per mg of total protein present in homogenates, at 25 1C and pH 7.00. ALT, AST, and LDH activities were measured with a colorimetric method employing commercial kits (Doles Reagents Ltda., Goiaˆnia, GO, Brazil). Preliminary tests showed extremely low activity of these enzymes in hemolymph (data not shown), which leads us to measure ALT, AST, and LDH activity in the hepatopancreas. It was considered that if microcystins induced damage in this organ, lower enzyme activities should be expected. Total protein content in the homogenate was determined using a commercial kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil), employing the biuret reagent. Determinations were done at 550 nm at least in duplicate. Lipid peroxides (LPO) were measured according to Hermes-Lima et al. (1995) and modified for microplate reader (Biotek ELx 800, Winooski, VT). Hepatopancreas samples were homogenized (9% W/V) in methanol 100%, and centrifuged at 1000g (4 1C) for 10 min and the supernatants kept for bioassay. LPO were determined using 90 mL of FeSO4 (1 mM), 35 mL of H2SO4 (0.25 mM), 35 mL of xylenol orange (1 mM, Sigma), 170 mL of MilliQ water, and 20 mL of the methanolic extract. All reagents were added following a sequential order mentioned above. Samples were incubated at room temperature until the reaction was completed (75 min), and then the absorbance (550 nm) was
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registered. Cumene hidroperoxide (CHP; 0.1 mM; Sigma) was employed as standard. Lipid peroxides were quantified in terms of CHP equivalents g1 of wet weight (ww) of tissue. Glycogen content was analyzed by homogenizing the hepatopancreas in citrate buffer (100 mM; pH 5.00) and then incubated with a-amyloglucosidase enzyme, according to the method described in Robaldo et al. (1999). Glucose content in samples was measured using a colorimetric method employing a commercial kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil). The results were expressed in terms of mg glucose g1 of wet weight (ww) of tissue. Data were subjected to a variance analysis (ANOVA) followed by the Newman–Keuls test (Zar, 1984). Normality and variance homogeneity were previously checked and the logarithmic transformation applied when needed. In all statistical tests, a significance level of 5% was adopted.
3. Results Oxygen consumption increased (po0:05) at a dose of 860 mg kg1 after 12 h of exposure (Fig. 1a). In bioassay 2 (72 h exposure), higher oxygen consumption (po0:05) was observed in all microcystins exposed groups (34, 172, or 860 mg kg1) (Fig. 1b). LPO levels remained unaltered (p40:05) in the shortterm exposure experiment (6 and 12 h), although after 12 h crabs from both experimental groups showed an increase (po0:05) in lipid peroxidation with respect to crabs sacrificed after 6 h (Fig. 2a). In the assay at 72 h, LPO was higher (po0:05) in microcystin doses equal to or higher than 34 mg kg1 (Fig. 2b). During the short-term exposure experiment (6 and 12 h), higher (po0:05) GST activity was registered in crabs exposed to toxin after 12 h (Fig. 3a), but their activity remained unaltered (p40:05) in the experiment at 72 h (Fig. 3b). ALT and AST activities remained unaltered (p40:05) in all experimental conditions (Figs. 4 and 5). On the other hand, higher LDH activity was observed after 12 h in the microcystin-exposed group (po0:05; Fig. 6a); however, no difference (p40:05) was observed in the 72 h exposure experiment for this enzyme (Fig. 6b). Glycogen content was similar (p40:05) in both experimental groups (control and microcystins exposed) in the short-term experiment (bioassay 1; Fig. 7a). However, glycogen content decreased (po0:05) in the experiment at 72 h in organisms exposed to doses equal to or higher than 172 mg kg1 (Fig. 7b). In bioassay 1, microcystin concentration in the hepatopancreas of C. granulatus was 1.8270.53 mg kg1 (in control group) and 13.1770.56 mg kg1 in crabs exposed to 860 mg kg1 of microcystins. In bioassay 2,
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Fig. 1. Oxygen consumption (mg O2 h1 mg tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 4). Similar letters mean absence of statistical differences (p40:05).
the concentration of microcystins in the control group and in the group exposed to 34 mg kg1 of microcystins was similar (p40:05; mean value: 5.6772.79 mg kg1), whereas no differences in microcystin content was registered in organisms exposed to 172 or 860 mg kg1 (mean value: 32.1474.12 mg kg1), being higher than in the control and 34 mg kg1 experimental groups.
4. Discussion The present study showed the induction of oxidative stress in crabs orally exposed to microcystins. This evidence came from the higher LPO levels found in organisms exposed to toxin during 72 h. Several factors can be responsible for this situation, including higher oxygen consumption promoting ROS generation (Yan and Sohal, 2000). However, it was not observed a linear relationship between oxygen consumption and LPO levels (see Figs. 1b and 2b). The stabilization of LPO content can be related to other causes, including
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Fig. 2. Lipid peroxides content (nmol of CHP g tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystin kg1 during 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05). CHP: cumene hydroperoxide, employed as standard.
antioxidant responses as reported in gills of the same species after microcystin exposure (Vinagre et al., 2003). It is also interesting to note that LPO levels were augmented after 12 h in both control and microcystins exposed groups respect 6 h (Fig. 2a). The same result was previously reported by Maciel et al. (2004). These authors found a daily variation in the levels of lipid peroxides in hepatopancreas of C. granulatus, which was lower at the beginning of the afternoon (when animals were sacrificed after 6 h of exposure, in the present study) and higher in the nocturnal period (when animals were sacrificed after 12 h of exposure, in the present study). Higher GST activity was observed after 12 h in the group exposed to microcystins, as previously reported by Gehringer et al. (2004a), who found augmented activities of GST between 8 and 16 h after a single dose of pure microcystin-LR administered to mice. A high GST activity can result from several factors, since this enzyme is an important component of the phase II conjugation system. Previous studies of Pflugmacher
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Fig. 3. Glutathione S-transferase (GST) activity (GST units mg proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
et al. (1998) showed that GST catalyzes the conjugation of microcystin with glutathione (GSH), and at this point a higher GST activity can be related to a higher detoxification capability in the hepatopancreas of crabs exposed to microcystins. On the other hand, GST also catalyzes the conjugation of GSH with oxidative products, such as 4-hydroxyalkenals (membrane peroxides) and/or base propenals, resulting from the DNA oxidative degradation (Leaver and George, 1998). Interestingly, the response in terms of GST was only observed in the short-term experiment (bioassay 1). In bioassay 2, higher levels of LPO were observed in crabs exposed to microcystins and a lack of GST response was verified. Studies of Srivastava et al. (1999) showed inhibition of m human isoenzyme GST by a metabolite of benzo[a]pyrene, indicating that some conjugation products catalyzed by GST may hinder this process. Other organic peroxides such as cumene hydroperoxide are known to inhibit a GST (Takamatsu and Inaba, 1994), meaning that oxidative products can in fact reduce the conjugation capabilities. The activity of the markers of hepatic damage (ALT, AST, and LDH) remained unaltered in bioassay 2, in
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Fig. 4. Alanine aminotransferase (ALT) activity (ALT units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
agreement with Gehringer et al. (2004b), who observed a lack of dose-dependent changes in ALT activity in mice exposed to microcystin-LR. Furthermore, Solter et al. (1998) reported that ALT is not a sensitive marker of subchronic exposure to microcystin-LR. Hepatopancreatic LDH increased after 12 h in crabs exposed to the toxins. This result was not expected, since the hepatopancreatic damage should be preceded by a decrease in LDH activity in hepatocites in parallel with an increase on the enzyme activity in the plasma. However, this was the only case where one of the markers of hepatic damage commonly employed in vertebrate species showed a significant response irrespective of the control group. Generally speaking, it seems that these biochemical measurements are not good markers of hepatopancreatic damage in invertebrate species, especially if compared with LPO and oxygen consumption responses. The glycogen content in hepatopancreas diminished in bioassay 2, suggesting that the higher oxygen consumption can be associated with the favored glycolitic pathway through activation of glycogen phosphorylase. An increase in the glucose metabolism
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can induce ROS (Dro¨ge, 2002), as previously shown in mice submitted to a chronic intake of diets with high glucose levels (Folmer et al., 2002). Microcystin content in bioassay 2 was higher than that found in bioassay 1, despite the same dose (860 mg kg1) being administered. This result suggests that C. granlatus is able to accumulate microcystins in its hepatopancreas, considering that the injections were administered at intervals of 24 h (so the crabs exposed for 72 h received three injections in total, and the crabs exposed for 6 and 12 h received a single oral dose). The results obtained in bioassay 2 indicated a dose–response relationship, since microcystins content increased in crabs exposed to the highest doses (172 and 860 mg of microcystins kg1) respect control crabs and those exposed to the lowest dose (34 mg of microcystins kg1). The fact that measurable levels of microcystins were detected in hepatopancreas of control can be explained by two hypothesis: (1) the existence of some compound(s) present in this tissue interfering in microcystins analysis, giving false-positive results (preliminaries HPLC studies showed the existence of compounds
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Fig. 5. Aspartate aminotransferase (AST) activity (AST units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins.kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
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Fig. 6. Lactato dehydrogenase (LDH) activity (LDH units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters means absence of statistical differences (p40:05).
which absorbance occurred at a wavelength similar to those absorbed by microcystin-LR); and (2) a previous pre-exposure of crabs to microcystins in their natural environment, previous to their collection. In this case, it can be considered that the elimination rate of microcystins from hepatopancreas of C. granulatus should be very low, since the acclimation period in the two bioassays was 1 month. Some differences were observed between the controls groups of the two bioassays performed. Organisms employed in bioassay 1 were collected in spring, whereas those employed in bioassay 2 were winter organisms. The fact that hepatopancreatic glycogen levels (see Fig. 7a and b) were lower in spring control organisms is in agreement with Kucharski and Silva (1991) that reported a similar decrease of glycogen in hepatopancreas of C. granulatus during this season. In addition with the seasonal variations, a stress factor(s) can be postulated to explain these variations, taking into account that crabs from bioassay 2 were more stressed than those of bioassay 1 because they received three injections and those from bioassay 1 received a single dose.
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and lower glycogen content in hepatopancreas), stressing the sensitivity of the hepatopancreas of C. granulatus to toxic stress induced by microcystins. Although the low internal doses of microcystin were enough to cause physiological and biochemical disturbances, the classical markers of hepatic damage of vertebrate species (ALT, AST, and LDH) remained unaltered in the long-term experiment, showing that these biochemical markers are not suitable to evidence hepatocellular injury in the estuarine crab Chasmagnathus granulatus. Finally, the elimination rate of microcystins in hepatopancreas of C. granulatus remains as an interesting topic to by analyzed in the future, through a depuration experiment.
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Acknowledgments
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Fig. 7. Glycogen content (mg glucose g tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 during 6 and 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
Conclusion The compensatory responses in the hepatopancreas of crabs exposed to microcystins showed a quick and transitory induction of GST activity that seems to be overwhelmed by the direct or indirect ROS generation induced by the toxins, as evidenced by the higher levels of LPO after 72 h. The accumulated microcystins in both bioassays showed an interesting feature in terms of the lower incorporation (13.1770.56 mg kg1 in crabs exposed to 860 mg kg1 in bioassay 1 and 32.147 4.12 mg kg1 in crabs exposed to doses equal of higher than 172 mg kg1 in bioassay 2). These values are so much lower if compared with other crustacean species like the crayfish Procambarus clarkii fed with toxic strains of Microcystis aeruginosa that accumulated 730 mg of toxin kg1 (Vasconcelos et al., 2001) and the prawn Penaeus monodon that accumulated 80 mg kg1 of microcystin (Kankaanpa¨a¨ et al., 2005). However even this low internal dose was sufficient to induce physiological (augmented oxygen consumption rates) and biochemical disturbances (mainly higher LPO levels
The authors are indebted to Fa´bio Maciel and Dr. Laura Geracitano for their support. Ligia J. Dewes and Juliana Z. Sandrini are graduate fellows from the Brazilian agency CNPq. Joa˜o S. Yunes and Jose´ M. Monserrat are research fellows from CNPq.
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xylenol orange complex formation. Free Radical Biol. Med. 19, 271–280. Kankaanpa¨a¨, H.T., Holliday, J., Schro¨der, H., Goddard, T.J., Fister, R.V., Carmichael, W.W., 2005. Cyanobacteria and prawn farming in Northern New South Wales, Australia—a case study on cyanobacteria diversity and hepatotoxin bioaccumulation. Toxicol. Appl. Pharmacol. 203 (3), 243–256. Kucharski, L.C.R., Silva, R.S.M., 1991. Seasonal variation in the energy metabolism in an estuarine crab, Chasmagnathus granulata (Dana, 1851). Comp. Biochem. Phyisiol. 100 (3), 599–602. Leaver, M.J., George, S.G., 1998. A piscine glutathione-S-transferase which efficiently conjugates the end-products of lipid peroxidation. Mar. Environ. Res. 46, 71–74. Maciel, F.E., Rosa, C.E., Santos, E.A., Monserrat, J.M., Nery, L.E.M., 2004. Daily variations in the oxygen consumption, antioxidant defenses and lipid peroxidation in gills and hepatopancreas of an estuarine crab, Chasmagnathus granulata (Decapoda, Brachyura). Can. J. Zool. Rev. Can. Zool. 82, 1871–1877. Malbrouck, C., Trausch, G., Devos, P., Kestemont, P., 2003. Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L. Comp. Biochem. Physiol. Part C 135, 39–48. Matthiensen, A., Beattie, K.A., Yunes, J.S., Kaya, K., CODD, G., 2000. [d-Leu1] Microcystin-LR, from the cyanobacteria Microcystis RST 9501 and from a Microcystis bloom from Patos Lagoon estuary, Brazil. Phytochemistry 55, 383–387. Minillo, A., Ferreira, A.H.F., Yogui, G.T., Yunes, J.S., 2000. Concentrac- o˜es de microcistinas e toxicidade nas formas coloniais de Microcystis aeruginosa de florac- o˜es no estua´rio da Lagoa dos Patos, RS. In: Espı´ ndola, E.L.G., Botta-Paschoal, C.M.R., Rocha, O., Bohrer, M.B.C., Oliveira-Neto, A.L. (Eds.), Ecotoxicologia: Perspectivas para o se´culo XXI. RiMa, Sa˜o Carlos, pp. 521–536. Montagnolli, W., Zamboni, A., Luvizotto-Santos, R., Yunes, J.S., 2004. Acute effects of Microcystis aeruginosa from the Patos Lagoon estuary, southern Brazil, on the microcrustacean Kalliapseudes schubartii (Crustacea: Tanaidacea). Arch. Environ. Contam. Toxicol. 46, 463–469. Nithart, M., Alliot, E., Salen-Picard, C., 1999. Production, respiration and ammonia excretion of two polychaete species in a north Norfolk saltmarsh. J. Mar. Biol. 79, 1029–1037. Pflugmacher, S., Wiegand, C., Oberemm, A., Beattie, K.A., Krause, E., Codd, G.A., Steinberg, C.E.W., 1998. Identification of an enzimaticaly formed glutathione conjugate of the cyanobacterial hepatoxin microcystin-LR: the first step of detoxication. Biochim. Biophys. Acta 1425, 527–533. Pinho, G.L.L., Rosa, C.M., Yunes, J.S., Luquet, C.M., Bianchini, A., Monserrat, J.M., 2003. Toxic effects of microcystins in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Comp. Biochem. Physiol. 135, 459–468. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., Stanier, R.Y., 1979. Generic assignments, strain histories and properties of pure culture of cyanobacteria. J. Gen. Microbiol. 111, 1–61.
Robaldo, R.B., Monserrat, J.M., Cousin, J.C.B., Bianchini, A., 1999. Influence of metacercariae (Digenea: Microphallidae) in the hepatopancreas of Chasmagnathus granulata (Decapoda: Grapsidae). Diseases Aquat. Organisms 37, 153–157. Runnegar, M., Berndt, N., Kaplowitz, N., 1995. Microcystin uptake and inhibition of protein phosphatases: Effects of chemoprotectants and self-inhibition in relation to know hepatic transporters. Toxicol. Appl. Pharmacol. 134, 264–272. Sies, H., 1999. Glutathione and its role in cellular functions. Free Radical Biol. Med. 27, 916–921. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water. A Guide to Their Public Health Consequences, Monitoring and Management. E&FN Spon, London, pp. 41–111. Solter, P.F., Wollenberg, G.K., Huang, X., Chu, F.S., Runnegar, M.T., 1998. Prolonged sublethal exposure to the protein phosphatase inhibitor microcystin-LR results in multiple dose-dependent hepatotoxic effects. Toxicol. Sci. 44, 87–96. Solter, P., Liu, Z., Guzman, R., 2000. Decreased hepatic ALT synthesis is an outcome of subchronic microcystin-LR toxicity. Toxicol. Appl. Pharmacol. 164, 216–220. Srivastava, S.K., Hu, X., Xia, H., Awasthi, S., Amin, S., Singh, S.V., 1999. Metabolic fate of glutathione conjugate of benzo[a]pyrene(7R, 8S)-diol (9S, 10R)-epoxide in human liver. Arch. Biochem. Biophys. 371, 340–344. Takamatsu, Y., Inaba, T., 1994. Inter-individual variability of human hepatic S-tranferase isozymes assessed by inhibitory capacity. Toxicology 88, 191–200. Vasconcelos, V., Oliveira, S., Teles, F.O., 2001. Impact of a toxic and a non-toxic strain of Microcystis aeruginosa on the crayfish Procambarus clarkii. Toxicon 39, 1461–1470. Vinagre, T.M., Alciati, J.C., Regoli, F., Bocchetti, R., Yunes, J.S., Bianchini, A., Monserrat, J.M., 2003. Effect of microcystin on ion regulation and antioxidant system in gills of the estuarine crab Chasmagnathus granulatus (Decapoda, Grapsidae). Comp. Biochem. Physiol. Part C 135, 67–75. Yan, L.-Y., Sohal, R.S., 2000. Prevention of flight activity prolongs the life span of the housefly, Musca domestica, and attenuates the ageassociated oxidative damage to specific mitochondrial proteins. Free Radical Biol. Med. 29, 1143–1150. Yunes, J.S., Niencheski, L.F.H., Salomon, P.S., Parise, M., Beattie, K.A., Ragget, S.L., Codd, G.A., 1998. Effect of nutrient balance and physical factors on blooms of toxic cyanobacteria in the Patos Lagoon, southern Brazil. Verhandl. Int. Verein. Theor. Angew. Limnol. 26, 1796–1800. Yunes, J.S., Monserrat, J.M., Lea˜o, J.C., Giordano, S., Pinho, G.L., Dewes, L.J., 2004. Is the environment also showing the impact of cyanotoxins? In: Abstracts, Sixth International Conference on Toxic Cyanobacteria, Bergen, Norway, p. 29. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ. Zˇegura, B., Sedmark, B., Filipicˇ, M., 2003. Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2. Toxicon 41, 41–48.
Ecotoxicology and Environmental Safety 65 (2006) 201–208 www.elsevier.com/locate/ecoenv
Biochemical and physiological responses after exposure to microcystins in the crab Chasmagnathus granulatus (Decapoda, Brachyura) L.J. Dewesa,b,, J.Z. Sandrinic,d, J.M. Monserratb,c,d, J.S. Yunesa,b Unidade de Pesquisa em Cianobacte´rias, Fundac- a˜o Universidade Federal do Rio Grande (FURG), Av. Ita´lia Km 8, Campus Carreiros, Hidroquı´mica, Caixa Postal 474, Rio Grande, RS, Brazil b Programa de Po´s-Graduac- a˜o em Oceanografia Biolo´gica, FURG, Brazil c Programa de Po´s-Graduac- a˜o em Cieˆncias Fisiolo´gicas, Fisiologia Animal Comparada, FURG, Brazil d Departamento de Cieˆncias Fisiolo´gicas, FURG, Brazil
a
Received 2 December 2004; received in revised form 8 July 2005; accepted 11 July 2005 Available online 31 August 2005
Abstract Microcystins are usually the predominant cyanotoxins present in both drinking and recreational waters after cyanobacterial blooms. Their classic toxic effect is hepatotoxicity through inhibition of serine/threonine phosphatases. However, recent studies also reported oxidative stress generation and disruption of ion regulation in aquatic organisms after microcystins exposure. In the present study, aqueous extracts of Microcystis aeruginosa were administered to the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) by gavage in variable doses (from 34 to 860 mg kg1) and exposure times (6, 12, and 72 h). A control group was exposed to saline solution. Analyzed variables included oxygen consumption, lipid peroxidation (LPO), enzyme activities (glutathione S-transferases or GST; alanine aminotransferase or ALT; aspartate aminotransferase or AST; and lactate dehydrogenase or LDH), glycogen, and microcystins content. Oxygen consumption increased in organisms exposed for 12h to 860 mg kg1 of microcystins and a similar result was observed after 72 h at doses equal to or higher than 34 mg kg1. LPO levels increased in doses equal to or higher than 34 mg kg1 after 72 h. GST and LDH activities increased after 12 h (at a dose of 860 mg kg1), but ALT and AST activities remained unaltered in all experimental conditions. Glycogen content decreased after 72 h exposure at doses equal to or higher than 172 mg kg1. After 12 h of exposure to 860 mg kg1 of microcystins, the concentration found in the hepatopancreas of C. granulatus was 13.1770.56 mg kg1. In crabs exposed to doses higher than 172 mg kg1 during 72 h this value raised to 32.1474.12 mg kg1. The obtained results indicated that microcystins exposure led the tissue to an oxidative stress condition (high LPO levels), at least in part favored by the augment of oxygen consumption, altering the glycogen metabolism. GST responses were only observed in the short-term experiment (12 h) and no effect on classical markers of vertebrate liver damage (ALT and AST) was observed. Although the hepatopancreas from C. granulatus accumulated a relatively low concentration of toxins, it was enough to induce physiological and biochemical disturbances. r 2005 Elsevier Inc. All rights reserved. Keywords: Microcystins; Chasmagnathus granulatus; Hepatopancreas; Glutathione S-transferases; Lipid peroxidation; Glycogen
1. Introduction The Patos Lagoon in southern Brazil is the second largest freshwater body in South America. Its waters are Corresponding author. Fax: +55 53 2336737.
E-mail address: [email protected] (L.J. Dewes). 0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2005.07.013
used for drinking, leisure, navigation, and agriculture by more than a million inhabitants. Furthermore, the lagoon estuarine region is a very rich and complex ecosystem with several fish, mollusk, and crustacea species being commercially exploited (Yunes et al., 2004). Toxic blooms of cyanobacteria, usually dominated by Microcystis aeruginosa, have been registered in
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the Patos Lagoon (RS, Brazil) in the past two decades, and the microcystins released in the water have reached concentrations as high as 289 mg L1 (Minillo et al., 2000). Microcystins are known to inhibit phosphatases of types 1 and 2A, resulting in excessive phosphorylation of cytoskeletal filaments, leading to liver failure (Carmichael, 1992). These toxins are cyclic heptapeptides, with a generic structure of cyclo-(D-Ala1-X2-DMeAsp3-Y4-Adda5-D-Glu6-Mdha7), where X and Y are variable L-aminoacids (Sivonen and Jones, 1999). [D-Leu1]Microcystin-LR was identified as the most abundant microcystin released from the strain of M. aeruginosa used in this work (RST 9501) (Matthiensen et al., 2000). The hepatic damage caused by microcystins can be measured by some clinical enzyme markers, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH). ALT is a transaminase that has been used as an important clinical indicator of hepatocellular injury (Solter et al., 2000). Recent evidence has shown that microcystins also induce oxidative stress and disruption of osmotic and ion regulation in crustacean species (Pinho et al., 2003; Vinagre et al., 2003). In vertebrates, microcystins are taken up into the hepatocytes by multispecific bile acid transporters (Eriksson et al., 1990; Runnegar et al., 1995), augmenting the production of reactive oxygen species (ROS), lipid peroxides, DNA damage, and activity of antioxidant enzymes (Ding et al., 1998, 2001; Gehringer et al., 2004a; Malbrouck et al., 2003; Zˇegura et al., 2003). The detoxification of microcystins in the liver is known to occur via conjugation to glutathione (GSH) by glutathione-S-transferase (GST) activity (Pflugmacher et al., 1998). It is important to note that GSH is considered one of the main nonenzymatic antioxidant and constitutes the first line of protection against ROS (Griffith, 1999; Sies, 1999). In this way, the conjugation of microcystin with GSH should represent a leak of this tripeptide from the intracellular pool, promoting the generation of oxidative stress. Microcystins still can promote oxidative stress through an increase of oxygen consumption, previously observed in aquatic organisms exposed to these toxins (Montagnolli et al., 2004), which in turn could increase ROS production. Augmented oxygen consumption can be related to a higher activity of the glycolitic pathway, through the stimulation of the enzyme glycogen phosphorylase via inhibition of protein phosphatases by microcystins. An increase of the glycolitic pathway can also promote ROS generation (Dro¨ge, 2002; Folmer et al., 2002). Up to date, little information exists about the internal concentration of microcystins in aquatic invertebrate organism after exposure to these kind of toxins and the specific effects on ALT, AST, and LDH activities. Taking into account this fact, the present study aimed to
evaluate the dose effects of microcystins in some biochemical and physiological parameters in the hepatopancreas of C. granulatus, including some related to oxidative stress as lipid peroxides levels.
2. Material and methods Adult male crabs were collected in salt marshes round the city of Rio Grande (RS, Southern Brazil, 301200 S–321100 S) in winter and spring. They were immediately transferred to the Department of Physiological Sciences (Fundac- a˜o Universidade Federal do Rio Grande, Brazil) and maintained in tanks with water at controlled temperature (20 1C) and salinity (2%), under constant aeration, for at least 1 month. Photoperiod was fixed at 12L:12D. Crabs were fed 3 days a week with ground beef. The water salinity was fixed to 2%, as previous evidence indicates that blooms of M. aeruginosa in Patos Lagoon occur in low salinities (Yunes et al., 1998). Cells of M. aeruginosa (strain RST 9501; Matthiensen et al., 2000) were cultured in BG11 medium (plus 8.82 mM of NaNO3) at 2571 1C (Rippka et al., 1979) and employed as source of microcystins. Cells were sonicated for 3 min in 100 Hz and centrifuged (10,000g, 10 min) at room temperature. The supernatant was collected and stored at 80 1C until use. Microcystins content of the extracts was determined using a commercial enzyme-linked immunoassay (ELISA) with polyclonal antibodies (EnviroLogix Inc., Portland, ME). After acclimation, crabs were employed in two different bioassays. In bioassay 1 (crabs collected in spring, mean weight 9.0672.76 g; n ¼ 69), organisms were exposed during 6 and 12 h to 860 mg kg1 of microcystins. In bioassay 2 (crabs collected in winter, mean weight 8.0272.44 g; n ¼ 69), crabs were exposed during 72 h to 34, 172, and 860 mg kg1 of microcystins. The different doses were obtained by diluting the stock solution of microcystins (obtained as described above) in crustacean saline solution (Vinagre et al., 2003). The microcystins exposed groups received 100 ml of the cyanobacterial extract by gavage, while the control groups were exposed only to crustacean saline solution. At the end of the bioassays, crabs were sacrificed and their hepatopancreas dissected immediately. Oxygen consumption was measured in samples of about 50 mg of tissue. Other pieces of hepatopancreas were stored at 80 1C for biochemical analysis. Crabs were not fed during the exposure period. Oxygen consumption was determined according to Nithart et al. (1999) through the determination of oxygen concentration in time 0 and 30 min postincubation of tissue at 20 1C in physiological solution
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plus 1 mM of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; Sigma). For analysis of enzyme activity (GST, ALT, AST, and LDH), microcystins, and protein content, the hepatopancreas was homogenized (20% w/v) in a buffer containing Tris base (20 mM), EDTA (1 mM), dithiothreitol (DTT 1 mM, Sigma), sucrose (0.5 mM), and KCl (150 mM), the pH being adjusted to 7.60. Samples were centrifuged at 9000g (4 1C) for 30 min and the supernatants employed as enzyme source. For microcystin analysis, after homogenization samples were sonicated (3 min, 100 Hz), centrifuged (10 min, 10,000g), and diluted with an equal volume of chloroform. The supernatants were collected and the microcystin content determined by immunoassay. Glutathione-S-transferase (GST) activity was measured according to Habig et al. (1974) and Habig and Jakoby (1981) by evaluating the conjugation of reduced glutathione (GSH; 1 mM; Sigma) with 1-chloro-2,4dinitro benzene (CDNB, 1 mM; Sigma) at 340 nm for 1 min. Aliquots of hepatopancreas homogenates were incubated (25 1C) in phosphate buffer 0.1 M at pH 7.00. An extinction coefficient of 9.6 mM1 cm1 was employed in order to calculate the moles of CDNB conjugated. Enzyme activity was calculated considering the total protein content in the homogenate. One GST unit was defined as the amount of enzyme necessary to conjugate 1 mmol of 1-chloro-2,4-dinitrobenzene (Sigma) per minute and per mg of total protein present in homogenates, at 25 1C and pH 7.00. ALT, AST, and LDH activities were measured with a colorimetric method employing commercial kits (Doles Reagents Ltda., Goiaˆnia, GO, Brazil). Preliminary tests showed extremely low activity of these enzymes in hemolymph (data not shown), which leads us to measure ALT, AST, and LDH activity in the hepatopancreas. It was considered that if microcystins induced damage in this organ, lower enzyme activities should be expected. Total protein content in the homogenate was determined using a commercial kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil), employing the biuret reagent. Determinations were done at 550 nm at least in duplicate. Lipid peroxides (LPO) were measured according to Hermes-Lima et al. (1995) and modified for microplate reader (Biotek ELx 800, Winooski, VT). Hepatopancreas samples were homogenized (9% W/V) in methanol 100%, and centrifuged at 1000g (4 1C) for 10 min and the supernatants kept for bioassay. LPO were determined using 90 mL of FeSO4 (1 mM), 35 mL of H2SO4 (0.25 mM), 35 mL of xylenol orange (1 mM, Sigma), 170 mL of MilliQ water, and 20 mL of the methanolic extract. All reagents were added following a sequential order mentioned above. Samples were incubated at room temperature until the reaction was completed (75 min), and then the absorbance (550 nm) was
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registered. Cumene hidroperoxide (CHP; 0.1 mM; Sigma) was employed as standard. Lipid peroxides were quantified in terms of CHP equivalents g1 of wet weight (ww) of tissue. Glycogen content was analyzed by homogenizing the hepatopancreas in citrate buffer (100 mM; pH 5.00) and then incubated with a-amyloglucosidase enzyme, according to the method described in Robaldo et al. (1999). Glucose content in samples was measured using a colorimetric method employing a commercial kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil). The results were expressed in terms of mg glucose g1 of wet weight (ww) of tissue. Data were subjected to a variance analysis (ANOVA) followed by the Newman–Keuls test (Zar, 1984). Normality and variance homogeneity were previously checked and the logarithmic transformation applied when needed. In all statistical tests, a significance level of 5% was adopted.
3. Results Oxygen consumption increased (po0:05) at a dose of 860 mg kg1 after 12 h of exposure (Fig. 1a). In bioassay 2 (72 h exposure), higher oxygen consumption (po0:05) was observed in all microcystins exposed groups (34, 172, or 860 mg kg1) (Fig. 1b). LPO levels remained unaltered (p40:05) in the shortterm exposure experiment (6 and 12 h), although after 12 h crabs from both experimental groups showed an increase (po0:05) in lipid peroxidation with respect to crabs sacrificed after 6 h (Fig. 2a). In the assay at 72 h, LPO was higher (po0:05) in microcystin doses equal to or higher than 34 mg kg1 (Fig. 2b). During the short-term exposure experiment (6 and 12 h), higher (po0:05) GST activity was registered in crabs exposed to toxin after 12 h (Fig. 3a), but their activity remained unaltered (p40:05) in the experiment at 72 h (Fig. 3b). ALT and AST activities remained unaltered (p40:05) in all experimental conditions (Figs. 4 and 5). On the other hand, higher LDH activity was observed after 12 h in the microcystin-exposed group (po0:05; Fig. 6a); however, no difference (p40:05) was observed in the 72 h exposure experiment for this enzyme (Fig. 6b). Glycogen content was similar (p40:05) in both experimental groups (control and microcystins exposed) in the short-term experiment (bioassay 1; Fig. 7a). However, glycogen content decreased (po0:05) in the experiment at 72 h in organisms exposed to doses equal to or higher than 172 mg kg1 (Fig. 7b). In bioassay 1, microcystin concentration in the hepatopancreas of C. granulatus was 1.8270.53 mg kg1 (in control group) and 13.1770.56 mg kg1 in crabs exposed to 860 mg kg1 of microcystins. In bioassay 2,
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Fig. 1. Oxygen consumption (mg O2 h1 mg tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 4). Similar letters mean absence of statistical differences (p40:05).
the concentration of microcystins in the control group and in the group exposed to 34 mg kg1 of microcystins was similar (p40:05; mean value: 5.6772.79 mg kg1), whereas no differences in microcystin content was registered in organisms exposed to 172 or 860 mg kg1 (mean value: 32.1474.12 mg kg1), being higher than in the control and 34 mg kg1 experimental groups.
4. Discussion The present study showed the induction of oxidative stress in crabs orally exposed to microcystins. This evidence came from the higher LPO levels found in organisms exposed to toxin during 72 h. Several factors can be responsible for this situation, including higher oxygen consumption promoting ROS generation (Yan and Sohal, 2000). However, it was not observed a linear relationship between oxygen consumption and LPO levels (see Figs. 1b and 2b). The stabilization of LPO content can be related to other causes, including
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Fig. 2. Lipid peroxides content (nmol of CHP g tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystin kg1 during 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05). CHP: cumene hydroperoxide, employed as standard.
antioxidant responses as reported in gills of the same species after microcystin exposure (Vinagre et al., 2003). It is also interesting to note that LPO levels were augmented after 12 h in both control and microcystins exposed groups respect 6 h (Fig. 2a). The same result was previously reported by Maciel et al. (2004). These authors found a daily variation in the levels of lipid peroxides in hepatopancreas of C. granulatus, which was lower at the beginning of the afternoon (when animals were sacrificed after 6 h of exposure, in the present study) and higher in the nocturnal period (when animals were sacrificed after 12 h of exposure, in the present study). Higher GST activity was observed after 12 h in the group exposed to microcystins, as previously reported by Gehringer et al. (2004a), who found augmented activities of GST between 8 and 16 h after a single dose of pure microcystin-LR administered to mice. A high GST activity can result from several factors, since this enzyme is an important component of the phase II conjugation system. Previous studies of Pflugmacher
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Fig. 3. Glutathione S-transferase (GST) activity (GST units mg proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
et al. (1998) showed that GST catalyzes the conjugation of microcystin with glutathione (GSH), and at this point a higher GST activity can be related to a higher detoxification capability in the hepatopancreas of crabs exposed to microcystins. On the other hand, GST also catalyzes the conjugation of GSH with oxidative products, such as 4-hydroxyalkenals (membrane peroxides) and/or base propenals, resulting from the DNA oxidative degradation (Leaver and George, 1998). Interestingly, the response in terms of GST was only observed in the short-term experiment (bioassay 1). In bioassay 2, higher levels of LPO were observed in crabs exposed to microcystins and a lack of GST response was verified. Studies of Srivastava et al. (1999) showed inhibition of m human isoenzyme GST by a metabolite of benzo[a]pyrene, indicating that some conjugation products catalyzed by GST may hinder this process. Other organic peroxides such as cumene hydroperoxide are known to inhibit a GST (Takamatsu and Inaba, 1994), meaning that oxidative products can in fact reduce the conjugation capabilities. The activity of the markers of hepatic damage (ALT, AST, and LDH) remained unaltered in bioassay 2, in
(b)
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Fig. 4. Alanine aminotransferase (ALT) activity (ALT units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
agreement with Gehringer et al. (2004b), who observed a lack of dose-dependent changes in ALT activity in mice exposed to microcystin-LR. Furthermore, Solter et al. (1998) reported that ALT is not a sensitive marker of subchronic exposure to microcystin-LR. Hepatopancreatic LDH increased after 12 h in crabs exposed to the toxins. This result was not expected, since the hepatopancreatic damage should be preceded by a decrease in LDH activity in hepatocites in parallel with an increase on the enzyme activity in the plasma. However, this was the only case where one of the markers of hepatic damage commonly employed in vertebrate species showed a significant response irrespective of the control group. Generally speaking, it seems that these biochemical measurements are not good markers of hepatopancreatic damage in invertebrate species, especially if compared with LPO and oxygen consumption responses. The glycogen content in hepatopancreas diminished in bioassay 2, suggesting that the higher oxygen consumption can be associated with the favored glycolitic pathway through activation of glycogen phosphorylase. An increase in the glucose metabolism
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can induce ROS (Dro¨ge, 2002), as previously shown in mice submitted to a chronic intake of diets with high glucose levels (Folmer et al., 2002). Microcystin content in bioassay 2 was higher than that found in bioassay 1, despite the same dose (860 mg kg1) being administered. This result suggests that C. granlatus is able to accumulate microcystins in its hepatopancreas, considering that the injections were administered at intervals of 24 h (so the crabs exposed for 72 h received three injections in total, and the crabs exposed for 6 and 12 h received a single oral dose). The results obtained in bioassay 2 indicated a dose–response relationship, since microcystins content increased in crabs exposed to the highest doses (172 and 860 mg of microcystins kg1) respect control crabs and those exposed to the lowest dose (34 mg of microcystins kg1). The fact that measurable levels of microcystins were detected in hepatopancreas of control can be explained by two hypothesis: (1) the existence of some compound(s) present in this tissue interfering in microcystins analysis, giving false-positive results (preliminaries HPLC studies showed the existence of compounds
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Fig. 5. Aspartate aminotransferase (AST) activity (AST units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins.kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
5.0
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Fig. 6. Lactato dehydrogenase (LDH) activity (LDH units g proteins1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 for 6 or 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters means absence of statistical differences (p40:05).
which absorbance occurred at a wavelength similar to those absorbed by microcystin-LR); and (2) a previous pre-exposure of crabs to microcystins in their natural environment, previous to their collection. In this case, it can be considered that the elimination rate of microcystins from hepatopancreas of C. granulatus should be very low, since the acclimation period in the two bioassays was 1 month. Some differences were observed between the controls groups of the two bioassays performed. Organisms employed in bioassay 1 were collected in spring, whereas those employed in bioassay 2 were winter organisms. The fact that hepatopancreatic glycogen levels (see Fig. 7a and b) were lower in spring control organisms is in agreement with Kucharski and Silva (1991) that reported a similar decrease of glycogen in hepatopancreas of C. granulatus during this season. In addition with the seasonal variations, a stress factor(s) can be postulated to explain these variations, taking into account that crabs from bioassay 2 were more stressed than those of bioassay 1 because they received three injections and those from bioassay 1 received a single dose.
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and lower glycogen content in hepatopancreas), stressing the sensitivity of the hepatopancreas of C. granulatus to toxic stress induced by microcystins. Although the low internal doses of microcystin were enough to cause physiological and biochemical disturbances, the classical markers of hepatic damage of vertebrate species (ALT, AST, and LDH) remained unaltered in the long-term experiment, showing that these biochemical markers are not suitable to evidence hepatocellular injury in the estuarine crab Chasmagnathus granulatus. Finally, the elimination rate of microcystins in hepatopancreas of C. granulatus remains as an interesting topic to by analyzed in the future, through a depuration experiment.
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Fig. 7. Glycogen content (mg glucose g tissue1) in hepatopancreas of the estuarine crab Chasmagnathus granulatus exposed to 860 mg of microcystins kg1 during 6 and 12 h (a) or after exposure to 34, 172, or 860 mg of microcystins kg1 for 72 h (b). Data are expressed as mean+1 standard deviation (n ¼ 5). Similar letters mean absence of statistical differences (p40:05).
Conclusion The compensatory responses in the hepatopancreas of crabs exposed to microcystins showed a quick and transitory induction of GST activity that seems to be overwhelmed by the direct or indirect ROS generation induced by the toxins, as evidenced by the higher levels of LPO after 72 h. The accumulated microcystins in both bioassays showed an interesting feature in terms of the lower incorporation (13.1770.56 mg kg1 in crabs exposed to 860 mg kg1 in bioassay 1 and 32.147 4.12 mg kg1 in crabs exposed to doses equal of higher than 172 mg kg1 in bioassay 2). These values are so much lower if compared with other crustacean species like the crayfish Procambarus clarkii fed with toxic strains of Microcystis aeruginosa that accumulated 730 mg of toxin kg1 (Vasconcelos et al., 2001) and the prawn Penaeus monodon that accumulated 80 mg kg1 of microcystin (Kankaanpa¨a¨ et al., 2005). However even this low internal dose was sufficient to induce physiological (augmented oxygen consumption rates) and biochemical disturbances (mainly higher LPO levels
The authors are indebted to Fa´bio Maciel and Dr. Laura Geracitano for their support. Ligia J. Dewes and Juliana Z. Sandrini are graduate fellows from the Brazilian agency CNPq. Joa˜o S. Yunes and Jose´ M. Monserrat are research fellows from CNPq.
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