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Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species
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Ecotoxicology and Environmental Safety 61 (2005) 353–360 www.elsevier.com/locate/ecoenv
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Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species G.L.L. Pinhoa,b, C. Moura da Rosaa, F.E. Maciela, A. Bianchinia,b, J.S. Yunesc, L.A.O. Proenc- ad, J.M. Monserrata,b, a
Departamento de Cieˆncias Fisiolo´gicas, Fundac- a˜o Universidade Federal do Rio Grande, R. Eng Alfredo Huch 475, 96201-900 Rio Grande, RS, Brazil Programa de Po´s-graduac- a˜o em Cieˆncias Fisiolo´gicas– Fisiologia Animal Comparada, Fundac- a˜o Universidade Federal do Rio Grande, 96201-900 Rio Grande, RS, Brazil c Unidade de Pesquisa em Cianobacte´rias, Fundac- a˜o Universidade Federal do Rio Grande, 96201-900 Rio Grande, RS, Brazil d Centro de Cieˆncias Tecnolo´gicas da Terra e do Mar, Universidade do Vale de Itajaı´, 88302-202 Santa Catarina, SC, Brazil
b
Received 29 April 2004; received in revised form 3 September 2004; accepted 29 November 2004 Available online 29 January 2005
Abstract Antioxidant responses and oxidative stress were evaluated in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) after oral microcystin administration. Responses were evaluated through antioxidant enzyme activities (catalase-(CAT), superoxide dismutase, glutathione-S-transferase- (GST)). Nonproteic sulfhydril (NP-SH) groups, oxygen consumption, lipid peroxides (LPO), and oxidized proteins were also measured. Microcystin administration increased the oxygen consumption. GST activity and NP-SH concentration showed transient increases and CAT activity showed a peak and then a reduction. Oxidative damage was evidenced with regard to LPO content and suggested by the inhibition of CAT activity at the end of the experiment, indicating that the antioxidant response induced by the toxin was insufficient. A lowering in the number of hepatopancreatic B cells should be related to microcystin elimination. r 2005 Elsevier Inc. All rights reserved. Keywords: Estuarine crab; Cyanobacteria; Microcystin; Oxidative stress; Lipid peroxidation; Catalase; Superoxide dismutase
1. Introduction It is known that the generation of the so-called reactive oxygen species (ROS) is a consequence of the appearance of aerobic life on earth. Thus, molecules such as hydrogen peroxide (H2O2), superoxide anion d (Od 2 ), and hydroxyl radical (HO ) constitute side effects of adopting an extremely efficient mechanism for energy generation through the oxidative metabolism pathway. In mammals, it has been estimated that almost 1–4% of the total oxygen consumption (OC) is converted into Od and H2O2 (Storey, 1996). The 2 Corresponding author. Departamento de Cieˆncias Fisiolo´gicas, Fundac- a˜o Universidade Federal do Rio Grande, R. Eng Alfredo Huch 475, 96201-900 Rio Grande, RS, Brasil. Fax: +55 53 233 6850. E-mail address: [email protected] (J.M. Monserrat).
0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.11.014
ROS generated must be intercepted or degraded to avoid oxidative damage to several macromolecules (protein, lipids, and DNA), a task accomplished by the antioxidant system (Halliwell and Gutteridge, 1999). This system includes enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), where the first two degrades Od 2 and the other two are involved in H2O2 degradation (Sies, 1993). An adjusted equilibrium between the concentration of prooxidant and that of antioxidant agents is a necessary condition to avoid oxidation of biomolecules by ROS. However, several chemicals can alter directly or indirectly this balance, favoring the generation of oxidative stress. Between these chemicals, hepatotoxins such as microcystins (MIC), produced by several genera of cyanobacteria including Microcystis and Anabaena,
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are believed to generate oxidative stress. The typical toxicological action of microcystins is due to serine/ threonine phosphatases inhibition, which leads to hyperphosphorylation of proteins, changes in cell cytoskeleton, disruption of cell signaling, etc. (Runnegar et al., 1995; Toivola and Eriksson, 1999). In recent years, new evidence has suggested that microcystin exposure also induces oxidative stress. Ding et al. (1998a, b) reported augmented ROS concentration after exposure of hepatocytes to microcystin. Ding et al. (1998a) showed that cultured hepatocytes presented higher levels of lipid peroxides (LPO) after microcystin exposure. Finally, several authors observed DNA damage after microcystin exposure (Rao and Bhattacharya, 1996; Rao et al., 1998; Ding et al., 1999). The study of Zˇegura et al. (2003) provides convincing evidence that microcystins cause oxidative DNA damage; this effect was reduced when cells were treated with a hydroxyl radical scavenger such as dimethyl sulfoxide. Pflugmacher et al. (1998) described for the first time the formation of a microcystin–GSH conjugate in a reaction catalyzed by the enzyme glutathione S-transferase (GST). The fact that the microcystin–GSH complex has a much lower inhibitory potency on phosphatases and a reduced toxic effect on the liver are evidence that this conjugation reaction represents a detoxification process (Metcalf et al., 2000). Authors such as Ding et al. (2000) have shown that microcystin induces cytotoxicity and cytoskeleton changes. They also reported that these effects are ameliorated when hepatocytes were pretreated with a GSH precursor such as N-acetylcystein. On the other hand, treatment with an inhibitor of GSH synthesis (buthionine (S,R)-sulfoximine) increased the susceptibility of hepatocytes to the toxin (Ding et al., 2000). Overall, these results point to the importance of GSH in the cell protection against microcystin toxicity. The present study analyzed the effects of an aqueous extract of the toxic strain RST9501 of Microcystis aeruginosa (Yunes et al., 1996; Matthiensen et al., 2000) in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura). Results obtained support the idea that microcystins induce antioxidant responses and oxidative stress. An omnivorous crab species was employed here due to its ecological relevance, since cyanobacteria are natural items of C. granulatus diet (D’Incao et al., 1990), and with the aim to gain information on the toxic effects of hepatotoxins in invertebrates.
2. Material and methods Adult male crabs of C. granulatus (mean weight: 13.4570.04 g; n ¼ 124) were collected in salt marshes,
near Rio Grande city (southern Brazil, 32 1S) in winter. Once in the laboratory, they were acclimated to water at 2% salinity for at least 1 month. Room temperature and photoperiod were fixed at 25 1C and 12 L:12 D, respectively. Crabs were fed three times a week with ground beef. Cells of M. aeruginosa were cultured in BG11 (+8.82 mM NaNO3) medium at 2571 1C (Rippka et al., 1979) and employed as toxin source. The cells were frozen and thawed three times and centrifuged (12,000g) at room temperature, for 10 min, as previously described (Vinagre et al., 2003). The supernatant was collected and stored at 20 1C. Microcystin content in the extracts was determined using a commercial enzyme-linked immunoassay with polyclonal antibodies (EnviroLogix Inc., Portland, ME). Crabs were divided into three groups: control (CTR), low microcystin (low MIC), and high microcystin (high MIC) concentration. The CTR group received daily oral injections (100 ml) of the same saline employed in the M. aeruginosa cell culture. Every day and for up 7 days, crabs from the low and high MIC groups received orally (100 ml) 14.31 and 71.54 ng of microcystin, which represented doses of 1.06 and 5.32 mg/kg/ day, respectively. Crabs were not fed during the exposure period. After 0, 2, and 7 days of exposure, crabs were cryoanesthesized and sacrificed and had their hepatopancreases dissected. Several pieces of hepatopancreas were stored in liquid nitrogen for biochemical measurements while others were either immediately used for OC determinations or fixed for histological analysis. OC was measured using small pieces of hepatopancreas (5075 mg) collected as described above. The tissue was incubated at 20 1C in saline solution (10 mM MgCl2, 355 mM NaCl, 16.6 mM CaCl2, 5 mM H3BO3, 10 mM KHCO3, 8 mM Na3C6H5O7 2H2O; pH adjusted to 7.6) containing 1 mM protease inhibitor phenylmethylsulfonyl fluoride (1 mM; Sigma). The oxygen concentration in the saline solution was measured at time zero and after 30 min of incubation using a portable oxymeter. Oxygen consumption OC was expressed as mg O2/h/mg of wet weigh of tissue. For antioxidant enzyme analysis, hepatopancreas was homogenized (1:4) in a cold (4 1C) buffer solution containing Tris base (20 mM), EDTA (1 mM), dithiothreitol (1 mM; Sigma), sucrose (500 mM), and KCl (150 mM), with pH adjusted to 7.6. Homogenates were centrifuged at 9000g (4 1C) for 30 min and the supernatants were employed as antioxidant enzymes source. All enzymatic determinations were done at least in duplicate. The activity of the enzyme catalase was analyzed according to Beutler (1975), measuring the initial rate of H2O2 (50 mM) decomposition at 240 nm. The results were expressed in CAT units/mg protein, where one unit
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is the amount of enzyme that hydrolyzes 1 mmol of H2O2 per minute and per milligram of protein at 30 1C and pH 8.0. Superoxide dismutase activity was determined according to McCord and Fridovich (1969). In this assay, superoxide anion is generated by the xanthine/xanthine oxidase system. The subsequent reduction of cytochrome c was monitored at 550 nm. Enzyme activity was expressed as SOD units, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per minute and per milligram of protein at 25 1C and pH 7.8. GST activity was measured by monitoring at 340 nm the formation of a conjugate between 1 mM GSH and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974; Habig and Jakoby, 1981). The results were expressed in GST units/mg protein, where one unit is defined as the amount of enzyme that conjugates 1 mmol of CDNB per minute and per milligram of protein at 25 1C and pH 7.4. For all the enzymatic assays the reagents were supplied by Sigma and Merck. For the measurement of nonproteic sulfhydril groups (NP-SH) the employed method was based on Sedlak and Lindsay (1968). Tissues were homogenized (1:20) in EDTA 0.02 M. Sulfhydril content in the samples was measured after deproteinization with trichloroacetic acid (50%). Nonproteic sulfhydril groups were detected using 5,5-dithiobis(2-nitrobenzoic acid) (Sigma). Absorbance readings (405 nm) were done using a microplate reader (Biotek Elx 800). NP-SH content was referred to the protein concentration of each homogenate prior to the deproteinization. Protein oxidation was determined by measuring the carbonyl content according to Levine et al. (1990) and Gabbianelli et al. (2002) using 2,4-dinitrophenylhydrazine, (Sigma). Absorbance was determined at 370 nm and results were expressed in nmol carbonyl content/mg protein using a molar absorption coefficient of 22,000/ M/cm. In all cases, total protein content in homogenates was determined using a commercial reagent kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil), based on the Biuret reagent. Determinations were done in triplicate at 550 nm. Lipid peroxides were measured according to HermesLima et al. (1995) and adapted for microplate reader (Biotek ELx 800; Winooski, VT). LPO were determined in 20 ml of methanolic extract, using 90 ml of FeSO4 (1 mM), 35 ml of H2SO4 (0.25 mM), 35 ml of xylenol orange (1 mM; Sigma), and 170 ml of MilliQ water. Absorbance was measured at 550 nm. Cumene hydroperoxide (CHP; 0.1 mM; Sigma) was employed as standard. Lipid peroxides were expressed as CHP equivalents/g of wet tissue.
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Histological studies were performed according to Pinho et al. (2003). Hepatopancreas samples were collected as previously described, fixed in cold Bouin solution for 24 h at 4 1C, and then transferred to 70% ethanol. Finally, tissue pieces were dehydrated and included in paraffin. Sections of 6 mm were stained with hematoxilin–eosin. Observations were done on a light microscope at 400 magnification. The number of B cells (involved in intracellular digestion) were counted in each tubule according to Saravana Bhavan and Geraldine (2000). The results were subjected to analysis of variance (ANOVA) followed by the Newman–Keuls multiple range test (Zar, 1984). Normality and homogeneity of variances were previously checked. Logarithmic transformation was applied if needed. In all statistical tests a significance level of 5% was adopted.
3. Results By the second day of exposure, the OC of hepatopancreas of crabs exposed to the high microcystin concentration (0.2070.02 mg O2/h/mg of tissue) was higher than that of the control group (0.117 0.01 mg O2/h/mg of tissue). After 1 week of exposure, crabs from both low MIC (0.1970.01 mg O2/h/mg of tissue) and high MIC (0.2870.03 mg O2/h/mg of tissue) groups showed significantly (Po0:05) higher oxygen consumption than crabs from the CTR group (0.1170.01 O2/h/mg of tissue) (Fig. 1). GST and CAT activities were significantly affected by microcystin exposure. At day 2, GST activity was µg O2/h/mg tissue 0.4
control low MIC
0.3
high MIC
0.2
0.1
0.0 0
2 Days
7
Fig. 1. Oxygen consumption in the hepatopancreas of Chasmagnathus granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of Microcystis aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 527); %means significantly different (Po0:05) from the respective control.
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significantly higher (Po0:05) in the low MIC group than in the CTR group (Fig. 2a). At the same time, CAT activity increased (Po0:05) more than 5.5-fold in crabs from the high MIC group (3.4370.67 U CAT/mg of
U GST/mg of proteins 0.04
control low MIC
0.03
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0.01
(a)
0.00
0
5
7
2 U CAT/mg of proteins
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3 2
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proteins) compared to that in the CTR group (0.6070.17 U CAT/mg of proteins). However, it was significantly (Po0:05) lower (1.1570.21 U CAT/mg of proteins) than that in the CTR group (3.207 0.48 U CAT/mg of proteins) after 1 week of exposure to the toxin (Fig. 2b). On the other hand, SOD activity was not significantly (P40:05) affected by microcystin exposure (Fig. 2c). At day 2, NP-SH concentration was significantly (Po0:05) higher in hepatopancreas from crabs of the low MIC group than that in the CTR group (Fig. 3). In the CTR group, no significant differences (P40:05) in GST activity and NP-SH concentration were observed over the experimental period. Also, in the control group, CAT activity was significantly (Po0:05) higher after 7 days (Fig. 2b) and SOD activity was significantly (Po0:05) lower at days 2 and 7 (2.4270.65 and 1.4070.30 U SOD/mg of proteins, respectively) than those at day 0 (5.2670.76 U SOD/mg of proteins) (Fig. 2c). Oxidative damage, evidenced with respect to LPO, was augmented (Po0:05) in the hepatopancreas of crabs from the high MIC group after 7 days of exposure in comparison to the CTR group (187.42716.03 and 142.6179.96 nmol of CHP/g of tissue, respectively) (Fig. 4a). In the CTR group, LPO content did not change over the experimental period (P40:05). No significant (P40:05) difference in protein carbonyl content was observed between experimental groups and/or exposure times (Fig. 4b). Finally, the number of B cells per tubule was significantly (Po0:05) lower in the hepatopancreas of crabs from the low and high MIC groups (Fig. 5).
U SOD/mg of proteins
7.5
µmoles of NP-SH/mg of proteins 0.075 5.0
control low MIC
+
0.050
2.5
high MIC
+ 0.025
0.0 (c)
0
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Fig. 2. Antioxidant enzyme activity in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. (a) Glutathione S-transferase (GST) activity; (b) catalase (CAT) activity; (c) superoxide dismutase (SOD) activity. In all cases, data are expressed as means +1 standard error (n ¼ 5212); %means significantly different (Po0:05) from the respective control; +means in the control group significantly different (Po0:05) from that at day 0.
0.000 0
2 Days
7
Fig. 3. Nonproteic sulfyhydril groups (NP-SH) in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/ kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 5210); %means significantly different (Po0:05) from the respective control.
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nmoles CHP/g of tissue 250
200
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B cells per tubule
20
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12
150
8
100
50
0
+
+
4
0 0
(a) 7.5
2 Days
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T0
control
low MIC
high MIC
Treatment Fig. 5. Number of B cells per tubule in hepatopancreas of C. granulatus before (day 0; T0) and after (day 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 8226); %means significantly different (Po0:05) from the control group; +means significantly different (Po0:05) from that at time zero (T0).
nmoles carbonyl/mg of proteins
5.0
2.5
0.0
0
(b)
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Fig. 4. Oxidative damage in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. (a) Lipid peroxides content, expressed in equivalents of cumene hydroperoxide (CHP). (b) Protein carbonyl content. In all cases, data are expressed as means +1 standard error (n ¼ 5214); %means significantly different (Po0:05) from the respective control.
4. Discussion In vertebrates, the liver is the main target organ for entry of cyanobacterial toxins (Carmichael, 1992). The hepatotoxicity of microcystins acting through phosphatase inhibition has been well studied (Thompson and Pace, 1992; Metcalf et al., 2000). However, alternative mechanisms of microcystin toxicity such as through oxidative stress generation have not been fully elucidated. In the present study, the highest dose of microcystin increased the hepatopancreas oxygen consumption rate after 2 days. At the end of this assay, both doses induced higher oxygen consumption rates, indicating that microcystin exposure can induce ROS generation simply
by augmenting aerobic metabolism. This result can be linked to phosphatase inhibition, favoring glycogen degradation over synthesis and increasing glucose utilization. Gehringer et al. (2003) observed reduced glycogen levels in liver of microcystin-exposed mice, suggesting a higher glycolitic rate, which is known to increase ROS generation (Dro¨ge, 2002). Antioxidant enzymes such as SOD, CAT, and GPx constitute the major defensive system against ROS (Sies, 1993). In the present study, hepatopancreas CAT activity increased in crabs exposed to the highest dose of microcystin for 2 days. This result indicates an activation of the antioxidant defensive system, indicating direct or indirect ROS generation after microcystin exposure. However, CAT activity in crabs exposed to the highest dose of M. aeruginosa aqueous extract was significantly reduced at the end of the experiment. Lankoff et al. (2002) showed that nodularin, a hepatoxin from Nodularia spumigena that inhibits phosphatase, also reduced CAT activity in mouse liver. The fact that we observed a lack of SOD response during microcystin exposure should favor CAT inhibition if ROS such as Od are being generated. Previous 2 studies reported that singlet oxygen, superoxide (Od 2 ), and peroxyl radicals are CAT inhibitors (Kono and Fridovich, 1982; Escobar et al., 1996). Other authors such as Bainy et al. (1996) also have found similar results when comparing CAT activity in gills of tilapia (Oreochromis niloticus) sampled in a polluted and in a reference site. Fish from the polluted site presented lower CAT activity, whereas gill SOD activity was
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similar in fish from both sites. These two facts should imply an exacerbation of tissue oxidative stress, since H2O2 production as a consequence of SOD activity remains stable but the capacity of H2O2 degradation was lower in fish from the polluted site due to its lower CAT activity. A similar situation can be postulated in the present study. GST is an important phase II enzyme that catalyzes the conjugation of reduced glutathione (GSH) to cellular components damaged by ROS attack, leading to their detoxification (Storey, 1996). Interestingly, Pflugmacher et al. (1998) described the formation of a microcystin–GSH conjugate in a reaction catalyzed by GST. Previous results from Pinho et al. (2003) indicated higher GST activity in hepatopancreas of C. granulatus after microcystin exposure. Taking into account the studies of Pflugmacher et al. (1998) and Beattie et al. (2003), a higher GST activity should imply a greater detoxification capacity through conjugation of microcystin with GSH. However, preliminary in vitro experiments in our laboratory using HPLC to detect microcystin–GSH conjugates were unsuccessful (data not shown). Thus, the higher GST activity observed in crabs exposed to the low dose of microcystin for 2 days should be related to the conjugation of oxidative products, such as 4-hydroxyalkenals (membrane peroxides) and/or base propenals resulting from the DNA oxidative degradation (Leaver and George, 1998). Results from the NP-SH concentration assessment in hepatopancreas of microcystin-exposed crabs were similar to those of GST activity, since NP-SH showed a peak at the second day of exposure only in the low dose of the toxin. At the end of the experiment, it was significantly reduced with respect to that of day 2 (Fig. 3). Ding et al. (2000) reported a similar biphasic response of an important NP-SH such as GSH in rat hepatocytes during microcystin exposure. They suggested that the first increase in intracellular GSH is probably a cellular response to protect against the toxic effect induced by microcystin. The conjugation of microcystin with GSH should trigger the synthesis of GSH. In turn GSH depletion would be related to cell membrane damage and consequent GSH efflux. If the concentrations of prooxidant agents exceed that of antioxidant agents, oxidative damage should be verified. In this context, the higher LPO levels observed in crabs exposed to the high dose of microcystin indicate that the crab defense system was overwhelmed. It is important to note that this condition was evident at the end of the experiment (day 7), where (1) oxygen consumption was higher, (2) CAT activity was lower, and (3) activities of antioxidant enzymes (GST and SOD) were not different from those in the control group. Ding et al. (1998a) and Guzman and Solter (1999) also showed augmented levels of LPO in rat hepatocytes after microcystin exposure.
Hepatopancreatic B cells are involved in intracellular digestion and express P-glycoprotein (P-gp) (Al-Mohanna and Nott, 1989; Ko¨hler et al., 1998). According to Ko¨hler et al. (1998), the P-gp system is involved in the transport of toxins from the cytoplasm to the lysosomal compartment for later elimination by senescence of B cells, being expelled into the digestive lumen. The fact that crabs exposed to microcystin showed a low number of B cells suggests an involvement of P-gp in the defensive mechanism of crab hepatopancreas against microcystins, leading to a decrement in B cell number. As verified in different studies, microcystin is highly toxic to several animal species, with values of LD50 varying approximately between 50 and 550 mg/kg (Tencalla et al., 1994; Yunes et al., 1996; Matthiensen et al., 2000). However, few studies have analyzed the in vivo biochemical responses after administration of sublethal doses of microcystin. For example, Guzman and Solter (1999) exposed rats during 28 days to doses of 16, 32, and 48 mg/kg/day of microcystin, verifying oxidative damage with respect to augmented LPO levels and histopatologies. In the present study, microcystin effects such as oxidative stress and antioxidant responses were observed when employing doses as low as 1.06 and 5.32 mg/kg/day and in a shorter period of time (7 days). Based on data reported by Vasconcelos et al. (2001), it can be estimated that the crayfish Procambarus clarkii accumulated approximately 0.73 mg of microcystin/kg of wet weight. In another study (Vasconcelos, 1995), it was determined that microcystin accumulation in the mussel Mytilus galloprovincialis was about 2.63 mg of microcystin/kg of wet weight. These two values are extremely high compared with the estimated total amount of microcystin administered to C. granulatus in the present study. In the present study, crabs were forced to ingest 14.31 or 71.54 mg of microcystin per day. Thus, the total amount of toxin administered to the crabs were 7.5 and 37.2 mg/kg of wet weight, respectively. These values are close to those determined by Magalha˜es et al. (2001) in muscle, liver, and viscera of the fish Tilapia randelli in areas where blooms of Microcystis sp. have occurred. Overall, these results suggest that toxic effects associated with oxidative damage must be determined simultaneously with determinations of toxin accumulation, if the objective is to evaluate deleterious effects of microcystin to aquatic fauna. In conclusion, the present results indicate that microcystin increases oxygen consumption, modifies enzymatic and nonenzymatic antioxidant defenses (CAT, GST, and NP-SH content), and induces oxidative damage with regard to LPO in different crab tissues. These results also indicate that oxidative stress is involved in toxicity of microcystin. Further studies should consider mechanisms of cellular absorption and elimination of microcystin to generate a better
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understanding of its mechanism of toxicity through oxidative stress generation in invertebrates.
Acknowledgments This research was supported by a grant from FAPERGS (Proc. No. 0021350) given to J.M. Monserrat. C. Moura da Rosa and F.E. Maciel are undergraduate fellows from FAPERGS and CNPq, respectively. G.L.L. Pinho is a graduate fellow from CAPES. A. Bianchini, J.M. Monserrat, and J.S. Yunes are research fellows from CNPq. References Al-Mohanna, S.Y., Nott, J.A., 1989. Functional cytology of the hepatopancreas of Penaeus simisulcatus (Crustacea: Decapoda) during the moult cycle. Mar. Biol. 101, 535–544. Bainy, A.C.D., Saito, E., Carvalho, P.S.M., Junqueira, V.B.C., 1996. Oxidative stress in gill, erythrocytes, liver and kidney of Nile tilapia (Oreochromis niloticus) from a polluted site. Aquat. Toxicol. 34, 151–162. Beattie, K.A., Ressler, J., Wiegand, C., Krause, E., Codd, G.A., Steinberg, C.E.W., Pflugmacher, S., 2003. Comparative effects and metabolism of two microcystins and nodularin in the brine shrimp Artemia salina. Aquat. Toxicol. 62, 219–226. Beutler, E., 1975. The preparation of red cells for assay. In: Beutler, E. (Ed.), Red Cell Metabolism: A Manual of Biochemical Methods. Grune & Straton, New York, pp. 8–18. Carmichael, W.W., 1992. Cyanobacterial secondary metabolites—the cyanotoxins. J. Appl. Bacteriol. 72, 445–459. D’Incao, F., Silva, K.G., Ruffino, M.L., Braga, A.C., 1990. Habito alimentar do caranguejo Chasmagnathus granulata Dana, 1851 na barra do Rio Grande, RS (Decapoda, Grapsidae). Atlaˆntica 12, 85–93. Ding, W.-X., Shen, H.M., Zhu, H.G., Ong, C.-N., 1998a. Studies on oxidative damage induced by cyanobacteria extract in primary cultured rat hepatocytes. Environ. Res. 78A, 12–18. Ding, W.-X., Shen, H.M., Zhu, H.G., Ong, C.-N., 1998b. Microcystic cyanobacteria causes mitochondrial membrane potential alteration and reactive species formation in primary cultured rat hepatocytes. Environ. Health Perspect. 106, 409–413. Ding, W.-X., Shen, H.M., Zhu, H.G., Lee, B.L., Ong, C.-N., 1999. Genotoxicity of microcystic cyanobacteria extract of a water source in China. Mut. Res. 442, 69–77. Ding, W.-X., Shen, H.M., Ong, C.-N., 2000. Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes. Environ. Health Perspect. 108, 605–609. Dro¨ge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95. Escobar, J.A., Rubio, M.A., Lissi, E.A., 1996. SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radic. Biol. Med. 20, 285–290. Gabbianelli, R., Falcioni, G., Nasuti, G., Cantalamessa, F., 2002. Cypermethrin-induced plasma membrane perturbation on erythrocytes from rats: reduction of fluidity in the hydrophobic core and in glutathione peroxidase activity. Toxicology 175, 91–101. Gehringer, M.M., Downs, K.S., Downing, T.G., Naude´, R.J., Shephard, E.G., 2003. An investigation into the effect of selenium supplementation on microcystin hepatotoxicity. Toxicon 41, 451–458.
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Guzman, R.E., Solter, P.F., 1999. Hepatic oxidative stress following prolonged sublethal microcystin LR exposure. Toxicol. Pathol. 27, 582–588. Habig, W.H., Jakoby, W.B., 1981. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 77, 398–405. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione-Stransferases. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Halliwell, B., Gutteridge, J.M., 1999. Free Radicals in Biology and Medicine. Oxford University Press, New York. Hermes-Lima, M., Willmore, W.G., Storey, K.B., 1995. Quantification of lipid peroxidation in tissue extracts based on Fe (III) xylenol orange complex formation. Free Radic. Biol. Med. 19, 271–280. Ko¨hler, A., Lauritzen, B., Jansen, D., Bottcher, P., Teguliwa, L., Krune, G., Broeg, K., 1998. Detection of P-glycoprotein mediated MDR/MXR in Carcinus maenas hepatopancreas by immuno-goldsilver labeling. Mar. Environ. Res. 46, 411–414. Kono, Y., Fridovich, I., 1982. Superoxide radical inhibits catalase. J. Biol. Chem. 10, 5751–5754. Lankoff, A., Bannasik, A., Nowak, M., 2002. Protective effects of melatonin against nodularin-induced oxidative stress in mouse liver. Arch. Toxicol. 76, 158–165. 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, 1–5. Levine, R.L., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.-G., Ahn, B.-W., Shaltiel, S., Stadtman, E.R., 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 186, 464–478. Magalha˜es, V.F., Soares, R.M., Azevedo, S.M.F., 2001. Microcystin contamination in fish from the Jacarepagua´ Lagoon (Rio de Janeiro, Brasil): ecological implication and human health risk. Toxicon 39, 1077–1085. Matthiensen, A., Beattie, K.A., Yunes, J.S., Kaya, K., Codd, G.A., 2000. [D-Leu1] microcistin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil. Phytochemistry 55, 383–387. McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function for erythocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6055. Metcalf, J.S., Beattie, K.A., Pflugmacher, S., Codd, G.A., 2000. Immuno-crossreactivity and toxicity assesment of conjugation products of the cyanobacterial toxin, microcystin-LR. FEMS Microbiol. Lett. 189, 155–158. 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., de 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. Rao, P.V.L., Bhattacharya, R., 1996. The cyanobacterial toxin microcystin-DNA damage in mouse liver in vivo. Toxicology 114, 29–36. Rao, P.V.L., Bhattacharya, R., Parida, M.M., Jana, A.M., Bhaskar, A.S.B., 1998. Freshwater cyanobacterium Microcystis aeruginosa (UTEX 2385) induced DNA damage in vivo an in vitro. Environ. Toxicol. Pharmacol. 5, 1–6. 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. Runnegar, M., Berndt, N., Kong, S., Lee, E.Y.C., Zhang, L., 1995. In vivo and in vitro binding of microcystin to protein phosphatases 1 and 2 A. Biochem. Biophys. Res. Commun. 216, 162–169.
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Saravana Bhavan, P., Geraldine, P., 2000. Histopathology of the hepatopancreas and gills of the prawn Macrobrachium malcolmsonii exposed to endosulfan. Aquat. Toxicol. 50, 331–339. Sedlak, J., Lindsay, R.H., 1968. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192–205. Sies, H., 1993. Strategies of antioxidant defense. Eur. J. Biochem. 215, 213–219. Storey, K.B., 1996. Oxidative stress: animal adaptations in nature. Braz. J. Med. Biol. Res. 29, 1715–1733. Tencalla, F.G., Dietrich, D.R., Schlatter, C., 1994. Toxicity of Microcystis aeruginosa peptide toxin to yearling rainbow trout (Oncorhyunchus mykiss). Aquat. Toxicol. 30, 215–224. Thompson, W.L., Pace, J.G., 1992. Substances that protect cultured hepatocytes from the toxic effects of microcystin-LR. Toxicol. In Vitro 6, 579–587. Toivola, D.M., Eriksson, J.E., 1999. Toxins affecting cell signalling and alteration of cytoskeletal structure. Toxicol. In Vitro 13, 521–530.
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. Vasconcelos, V.M., 1995. Uptake and depuration of the heptapeptide toxin microcystin-LR in Mytillus galloprovincialis. Aquat. Toxicol. 32, 227–237. Vinagre, T.M., Alciati, J.C., Regoli, F., Bocchetti, R., Yunes, J.S., Bianchini, A., Monserrat, J.M., 2003. Effect of hepatotoxins (microcystin) on ion-regulation and antioxidant system in gills of Chasmagnathus granulatus (Decapoda, Grapsidae). Comp. Biochem. Physiol. 135, 67–75. Yunes, J.S., Matthiensen, A., Salomon, P.S., Beattie, K., Raggett, S.L., Codd, G.A., 1996. Toxic blooms of cyanobacteria in the Patos Lagoon, RS. J. Aquat. Ecosys. Health 5, 223–229. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, New Jersey. 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 61 (2005) 353–360 www.elsevier.com/locate/ecoenv
Rapid communication
Antioxidant responses and oxidative stress after microcystin exposure in the hepatopancreas of an estuarine crab species G.L.L. Pinhoa,b, C. Moura da Rosaa, F.E. Maciela, A. Bianchinia,b, J.S. Yunesc, L.A.O. Proenc- ad, J.M. Monserrata,b, a
Departamento de Cieˆncias Fisiolo´gicas, Fundac- a˜o Universidade Federal do Rio Grande, R. Eng Alfredo Huch 475, 96201-900 Rio Grande, RS, Brazil Programa de Po´s-graduac- a˜o em Cieˆncias Fisiolo´gicas– Fisiologia Animal Comparada, Fundac- a˜o Universidade Federal do Rio Grande, 96201-900 Rio Grande, RS, Brazil c Unidade de Pesquisa em Cianobacte´rias, Fundac- a˜o Universidade Federal do Rio Grande, 96201-900 Rio Grande, RS, Brazil d Centro de Cieˆncias Tecnolo´gicas da Terra e do Mar, Universidade do Vale de Itajaı´, 88302-202 Santa Catarina, SC, Brazil
b
Received 29 April 2004; received in revised form 3 September 2004; accepted 29 November 2004 Available online 29 January 2005
Abstract Antioxidant responses and oxidative stress were evaluated in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura) after oral microcystin administration. Responses were evaluated through antioxidant enzyme activities (catalase-(CAT), superoxide dismutase, glutathione-S-transferase- (GST)). Nonproteic sulfhydril (NP-SH) groups, oxygen consumption, lipid peroxides (LPO), and oxidized proteins were also measured. Microcystin administration increased the oxygen consumption. GST activity and NP-SH concentration showed transient increases and CAT activity showed a peak and then a reduction. Oxidative damage was evidenced with regard to LPO content and suggested by the inhibition of CAT activity at the end of the experiment, indicating that the antioxidant response induced by the toxin was insufficient. A lowering in the number of hepatopancreatic B cells should be related to microcystin elimination. r 2005 Elsevier Inc. All rights reserved. Keywords: Estuarine crab; Cyanobacteria; Microcystin; Oxidative stress; Lipid peroxidation; Catalase; Superoxide dismutase
1. Introduction It is known that the generation of the so-called reactive oxygen species (ROS) is a consequence of the appearance of aerobic life on earth. Thus, molecules such as hydrogen peroxide (H2O2), superoxide anion d (Od 2 ), and hydroxyl radical (HO ) constitute side effects of adopting an extremely efficient mechanism for energy generation through the oxidative metabolism pathway. In mammals, it has been estimated that almost 1–4% of the total oxygen consumption (OC) is converted into Od and H2O2 (Storey, 1996). The 2 Corresponding author. Departamento de Cieˆncias Fisiolo´gicas, Fundac- a˜o Universidade Federal do Rio Grande, R. Eng Alfredo Huch 475, 96201-900 Rio Grande, RS, Brasil. Fax: +55 53 233 6850. E-mail address: [email protected] (J.M. Monserrat).
0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2004.11.014
ROS generated must be intercepted or degraded to avoid oxidative damage to several macromolecules (protein, lipids, and DNA), a task accomplished by the antioxidant system (Halliwell and Gutteridge, 1999). This system includes enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), where the first two degrades Od 2 and the other two are involved in H2O2 degradation (Sies, 1993). An adjusted equilibrium between the concentration of prooxidant and that of antioxidant agents is a necessary condition to avoid oxidation of biomolecules by ROS. However, several chemicals can alter directly or indirectly this balance, favoring the generation of oxidative stress. Between these chemicals, hepatotoxins such as microcystins (MIC), produced by several genera of cyanobacteria including Microcystis and Anabaena,
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are believed to generate oxidative stress. The typical toxicological action of microcystins is due to serine/ threonine phosphatases inhibition, which leads to hyperphosphorylation of proteins, changes in cell cytoskeleton, disruption of cell signaling, etc. (Runnegar et al., 1995; Toivola and Eriksson, 1999). In recent years, new evidence has suggested that microcystin exposure also induces oxidative stress. Ding et al. (1998a, b) reported augmented ROS concentration after exposure of hepatocytes to microcystin. Ding et al. (1998a) showed that cultured hepatocytes presented higher levels of lipid peroxides (LPO) after microcystin exposure. Finally, several authors observed DNA damage after microcystin exposure (Rao and Bhattacharya, 1996; Rao et al., 1998; Ding et al., 1999). The study of Zˇegura et al. (2003) provides convincing evidence that microcystins cause oxidative DNA damage; this effect was reduced when cells were treated with a hydroxyl radical scavenger such as dimethyl sulfoxide. Pflugmacher et al. (1998) described for the first time the formation of a microcystin–GSH conjugate in a reaction catalyzed by the enzyme glutathione S-transferase (GST). The fact that the microcystin–GSH complex has a much lower inhibitory potency on phosphatases and a reduced toxic effect on the liver are evidence that this conjugation reaction represents a detoxification process (Metcalf et al., 2000). Authors such as Ding et al. (2000) have shown that microcystin induces cytotoxicity and cytoskeleton changes. They also reported that these effects are ameliorated when hepatocytes were pretreated with a GSH precursor such as N-acetylcystein. On the other hand, treatment with an inhibitor of GSH synthesis (buthionine (S,R)-sulfoximine) increased the susceptibility of hepatocytes to the toxin (Ding et al., 2000). Overall, these results point to the importance of GSH in the cell protection against microcystin toxicity. The present study analyzed the effects of an aqueous extract of the toxic strain RST9501 of Microcystis aeruginosa (Yunes et al., 1996; Matthiensen et al., 2000) in the hepatopancreas of the estuarine crab Chasmagnathus granulatus (Decapoda, Brachyura). Results obtained support the idea that microcystins induce antioxidant responses and oxidative stress. An omnivorous crab species was employed here due to its ecological relevance, since cyanobacteria are natural items of C. granulatus diet (D’Incao et al., 1990), and with the aim to gain information on the toxic effects of hepatotoxins in invertebrates.
2. Material and methods Adult male crabs of C. granulatus (mean weight: 13.4570.04 g; n ¼ 124) were collected in salt marshes,
near Rio Grande city (southern Brazil, 32 1S) in winter. Once in the laboratory, they were acclimated to water at 2% salinity for at least 1 month. Room temperature and photoperiod were fixed at 25 1C and 12 L:12 D, respectively. Crabs were fed three times a week with ground beef. Cells of M. aeruginosa were cultured in BG11 (+8.82 mM NaNO3) medium at 2571 1C (Rippka et al., 1979) and employed as toxin source. The cells were frozen and thawed three times and centrifuged (12,000g) at room temperature, for 10 min, as previously described (Vinagre et al., 2003). The supernatant was collected and stored at 20 1C. Microcystin content in the extracts was determined using a commercial enzyme-linked immunoassay with polyclonal antibodies (EnviroLogix Inc., Portland, ME). Crabs were divided into three groups: control (CTR), low microcystin (low MIC), and high microcystin (high MIC) concentration. The CTR group received daily oral injections (100 ml) of the same saline employed in the M. aeruginosa cell culture. Every day and for up 7 days, crabs from the low and high MIC groups received orally (100 ml) 14.31 and 71.54 ng of microcystin, which represented doses of 1.06 and 5.32 mg/kg/ day, respectively. Crabs were not fed during the exposure period. After 0, 2, and 7 days of exposure, crabs were cryoanesthesized and sacrificed and had their hepatopancreases dissected. Several pieces of hepatopancreas were stored in liquid nitrogen for biochemical measurements while others were either immediately used for OC determinations or fixed for histological analysis. OC was measured using small pieces of hepatopancreas (5075 mg) collected as described above. The tissue was incubated at 20 1C in saline solution (10 mM MgCl2, 355 mM NaCl, 16.6 mM CaCl2, 5 mM H3BO3, 10 mM KHCO3, 8 mM Na3C6H5O7 2H2O; pH adjusted to 7.6) containing 1 mM protease inhibitor phenylmethylsulfonyl fluoride (1 mM; Sigma). The oxygen concentration in the saline solution was measured at time zero and after 30 min of incubation using a portable oxymeter. Oxygen consumption OC was expressed as mg O2/h/mg of wet weigh of tissue. For antioxidant enzyme analysis, hepatopancreas was homogenized (1:4) in a cold (4 1C) buffer solution containing Tris base (20 mM), EDTA (1 mM), dithiothreitol (1 mM; Sigma), sucrose (500 mM), and KCl (150 mM), with pH adjusted to 7.6. Homogenates were centrifuged at 9000g (4 1C) for 30 min and the supernatants were employed as antioxidant enzymes source. All enzymatic determinations were done at least in duplicate. The activity of the enzyme catalase was analyzed according to Beutler (1975), measuring the initial rate of H2O2 (50 mM) decomposition at 240 nm. The results were expressed in CAT units/mg protein, where one unit
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is the amount of enzyme that hydrolyzes 1 mmol of H2O2 per minute and per milligram of protein at 30 1C and pH 8.0. Superoxide dismutase activity was determined according to McCord and Fridovich (1969). In this assay, superoxide anion is generated by the xanthine/xanthine oxidase system. The subsequent reduction of cytochrome c was monitored at 550 nm. Enzyme activity was expressed as SOD units, where one unit is defined as the amount of enzyme needed to inhibit 50% of cytochrome c reduction per minute and per milligram of protein at 25 1C and pH 7.8. GST activity was measured by monitoring at 340 nm the formation of a conjugate between 1 mM GSH and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) (Habig et al., 1974; Habig and Jakoby, 1981). The results were expressed in GST units/mg protein, where one unit is defined as the amount of enzyme that conjugates 1 mmol of CDNB per minute and per milligram of protein at 25 1C and pH 7.4. For all the enzymatic assays the reagents were supplied by Sigma and Merck. For the measurement of nonproteic sulfhydril groups (NP-SH) the employed method was based on Sedlak and Lindsay (1968). Tissues were homogenized (1:20) in EDTA 0.02 M. Sulfhydril content in the samples was measured after deproteinization with trichloroacetic acid (50%). Nonproteic sulfhydril groups were detected using 5,5-dithiobis(2-nitrobenzoic acid) (Sigma). Absorbance readings (405 nm) were done using a microplate reader (Biotek Elx 800). NP-SH content was referred to the protein concentration of each homogenate prior to the deproteinization. Protein oxidation was determined by measuring the carbonyl content according to Levine et al. (1990) and Gabbianelli et al. (2002) using 2,4-dinitrophenylhydrazine, (Sigma). Absorbance was determined at 370 nm and results were expressed in nmol carbonyl content/mg protein using a molar absorption coefficient of 22,000/ M/cm. In all cases, total protein content in homogenates was determined using a commercial reagent kit (Doles Reagents Ltda., Goiaˆnia, GO, Brazil), based on the Biuret reagent. Determinations were done in triplicate at 550 nm. Lipid peroxides were measured according to HermesLima et al. (1995) and adapted for microplate reader (Biotek ELx 800; Winooski, VT). LPO were determined in 20 ml of methanolic extract, using 90 ml of FeSO4 (1 mM), 35 ml of H2SO4 (0.25 mM), 35 ml of xylenol orange (1 mM; Sigma), and 170 ml of MilliQ water. Absorbance was measured at 550 nm. Cumene hydroperoxide (CHP; 0.1 mM; Sigma) was employed as standard. Lipid peroxides were expressed as CHP equivalents/g of wet tissue.
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Histological studies were performed according to Pinho et al. (2003). Hepatopancreas samples were collected as previously described, fixed in cold Bouin solution for 24 h at 4 1C, and then transferred to 70% ethanol. Finally, tissue pieces were dehydrated and included in paraffin. Sections of 6 mm were stained with hematoxilin–eosin. Observations were done on a light microscope at 400 magnification. The number of B cells (involved in intracellular digestion) were counted in each tubule according to Saravana Bhavan and Geraldine (2000). The results were subjected to analysis of variance (ANOVA) followed by the Newman–Keuls multiple range test (Zar, 1984). Normality and homogeneity of variances were previously checked. Logarithmic transformation was applied if needed. In all statistical tests a significance level of 5% was adopted.
3. Results By the second day of exposure, the OC of hepatopancreas of crabs exposed to the high microcystin concentration (0.2070.02 mg O2/h/mg of tissue) was higher than that of the control group (0.117 0.01 mg O2/h/mg of tissue). After 1 week of exposure, crabs from both low MIC (0.1970.01 mg O2/h/mg of tissue) and high MIC (0.2870.03 mg O2/h/mg of tissue) groups showed significantly (Po0:05) higher oxygen consumption than crabs from the CTR group (0.1170.01 O2/h/mg of tissue) (Fig. 1). GST and CAT activities were significantly affected by microcystin exposure. At day 2, GST activity was µg O2/h/mg tissue 0.4
control low MIC
0.3
high MIC
0.2
0.1
0.0 0
2 Days
7
Fig. 1. Oxygen consumption in the hepatopancreas of Chasmagnathus granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of Microcystis aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 527); %means significantly different (Po0:05) from the respective control.
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significantly higher (Po0:05) in the low MIC group than in the CTR group (Fig. 2a). At the same time, CAT activity increased (Po0:05) more than 5.5-fold in crabs from the high MIC group (3.4370.67 U CAT/mg of
U GST/mg of proteins 0.04
control low MIC
0.03
high MIC
0.02
0.01
(a)
0.00
0
5
7
2 U CAT/mg of proteins
4
+
3 2
1
(b)
0
0
7
2
proteins) compared to that in the CTR group (0.6070.17 U CAT/mg of proteins). However, it was significantly (Po0:05) lower (1.1570.21 U CAT/mg of proteins) than that in the CTR group (3.207 0.48 U CAT/mg of proteins) after 1 week of exposure to the toxin (Fig. 2b). On the other hand, SOD activity was not significantly (P40:05) affected by microcystin exposure (Fig. 2c). At day 2, NP-SH concentration was significantly (Po0:05) higher in hepatopancreas from crabs of the low MIC group than that in the CTR group (Fig. 3). In the CTR group, no significant differences (P40:05) in GST activity and NP-SH concentration were observed over the experimental period. Also, in the control group, CAT activity was significantly (Po0:05) higher after 7 days (Fig. 2b) and SOD activity was significantly (Po0:05) lower at days 2 and 7 (2.4270.65 and 1.4070.30 U SOD/mg of proteins, respectively) than those at day 0 (5.2670.76 U SOD/mg of proteins) (Fig. 2c). Oxidative damage, evidenced with respect to LPO, was augmented (Po0:05) in the hepatopancreas of crabs from the high MIC group after 7 days of exposure in comparison to the CTR group (187.42716.03 and 142.6179.96 nmol of CHP/g of tissue, respectively) (Fig. 4a). In the CTR group, LPO content did not change over the experimental period (P40:05). No significant (P40:05) difference in protein carbonyl content was observed between experimental groups and/or exposure times (Fig. 4b). Finally, the number of B cells per tubule was significantly (Po0:05) lower in the hepatopancreas of crabs from the low and high MIC groups (Fig. 5).
U SOD/mg of proteins
7.5
µmoles of NP-SH/mg of proteins 0.075 5.0
control low MIC
+
0.050
2.5
high MIC
+ 0.025
0.0 (c)
0
2 Days
7
Fig. 2. Antioxidant enzyme activity in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. (a) Glutathione S-transferase (GST) activity; (b) catalase (CAT) activity; (c) superoxide dismutase (SOD) activity. In all cases, data are expressed as means +1 standard error (n ¼ 5212); %means significantly different (Po0:05) from the respective control; +means in the control group significantly different (Po0:05) from that at day 0.
0.000 0
2 Days
7
Fig. 3. Nonproteic sulfyhydril groups (NP-SH) in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/ kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 5210); %means significantly different (Po0:05) from the respective control.
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nmoles CHP/g of tissue 250
200
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B cells per tubule
20
control
low MIC high MIC
16
12
150
8
100
50
0
+
+
4
0 0
(a) 7.5
2 Days
7
T0
control
low MIC
high MIC
Treatment Fig. 5. Number of B cells per tubule in hepatopancreas of C. granulatus before (day 0; T0) and after (day 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. In all cases, data are expressed as means +1 standard error (n ¼ 8226); %means significantly different (Po0:05) from the control group; +means significantly different (Po0:05) from that at time zero (T0).
nmoles carbonyl/mg of proteins
5.0
2.5
0.0
0
(b)
2 Days
7
Fig. 4. Oxidative damage in the hepatopancreas of C. granulatus before (day 0) and after (days 2 and 7) oral injection (100 ml) of saline solution (control), low dose (1.06 mg of microcystins/kg/day; low MIC), or high dose (5.32 mg of microcystins/kg/day; high MIC) of an aqueous extract of M. aeruginosa. (a) Lipid peroxides content, expressed in equivalents of cumene hydroperoxide (CHP). (b) Protein carbonyl content. In all cases, data are expressed as means +1 standard error (n ¼ 5214); %means significantly different (Po0:05) from the respective control.
4. Discussion In vertebrates, the liver is the main target organ for entry of cyanobacterial toxins (Carmichael, 1992). The hepatotoxicity of microcystins acting through phosphatase inhibition has been well studied (Thompson and Pace, 1992; Metcalf et al., 2000). However, alternative mechanisms of microcystin toxicity such as through oxidative stress generation have not been fully elucidated. In the present study, the highest dose of microcystin increased the hepatopancreas oxygen consumption rate after 2 days. At the end of this assay, both doses induced higher oxygen consumption rates, indicating that microcystin exposure can induce ROS generation simply
by augmenting aerobic metabolism. This result can be linked to phosphatase inhibition, favoring glycogen degradation over synthesis and increasing glucose utilization. Gehringer et al. (2003) observed reduced glycogen levels in liver of microcystin-exposed mice, suggesting a higher glycolitic rate, which is known to increase ROS generation (Dro¨ge, 2002). Antioxidant enzymes such as SOD, CAT, and GPx constitute the major defensive system against ROS (Sies, 1993). In the present study, hepatopancreas CAT activity increased in crabs exposed to the highest dose of microcystin for 2 days. This result indicates an activation of the antioxidant defensive system, indicating direct or indirect ROS generation after microcystin exposure. However, CAT activity in crabs exposed to the highest dose of M. aeruginosa aqueous extract was significantly reduced at the end of the experiment. Lankoff et al. (2002) showed that nodularin, a hepatoxin from Nodularia spumigena that inhibits phosphatase, also reduced CAT activity in mouse liver. The fact that we observed a lack of SOD response during microcystin exposure should favor CAT inhibition if ROS such as Od are being generated. Previous 2 studies reported that singlet oxygen, superoxide (Od 2 ), and peroxyl radicals are CAT inhibitors (Kono and Fridovich, 1982; Escobar et al., 1996). Other authors such as Bainy et al. (1996) also have found similar results when comparing CAT activity in gills of tilapia (Oreochromis niloticus) sampled in a polluted and in a reference site. Fish from the polluted site presented lower CAT activity, whereas gill SOD activity was
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similar in fish from both sites. These two facts should imply an exacerbation of tissue oxidative stress, since H2O2 production as a consequence of SOD activity remains stable but the capacity of H2O2 degradation was lower in fish from the polluted site due to its lower CAT activity. A similar situation can be postulated in the present study. GST is an important phase II enzyme that catalyzes the conjugation of reduced glutathione (GSH) to cellular components damaged by ROS attack, leading to their detoxification (Storey, 1996). Interestingly, Pflugmacher et al. (1998) described the formation of a microcystin–GSH conjugate in a reaction catalyzed by GST. Previous results from Pinho et al. (2003) indicated higher GST activity in hepatopancreas of C. granulatus after microcystin exposure. Taking into account the studies of Pflugmacher et al. (1998) and Beattie et al. (2003), a higher GST activity should imply a greater detoxification capacity through conjugation of microcystin with GSH. However, preliminary in vitro experiments in our laboratory using HPLC to detect microcystin–GSH conjugates were unsuccessful (data not shown). Thus, the higher GST activity observed in crabs exposed to the low dose of microcystin for 2 days should be related to the conjugation of oxidative products, such as 4-hydroxyalkenals (membrane peroxides) and/or base propenals resulting from the DNA oxidative degradation (Leaver and George, 1998). Results from the NP-SH concentration assessment in hepatopancreas of microcystin-exposed crabs were similar to those of GST activity, since NP-SH showed a peak at the second day of exposure only in the low dose of the toxin. At the end of the experiment, it was significantly reduced with respect to that of day 2 (Fig. 3). Ding et al. (2000) reported a similar biphasic response of an important NP-SH such as GSH in rat hepatocytes during microcystin exposure. They suggested that the first increase in intracellular GSH is probably a cellular response to protect against the toxic effect induced by microcystin. The conjugation of microcystin with GSH should trigger the synthesis of GSH. In turn GSH depletion would be related to cell membrane damage and consequent GSH efflux. If the concentrations of prooxidant agents exceed that of antioxidant agents, oxidative damage should be verified. In this context, the higher LPO levels observed in crabs exposed to the high dose of microcystin indicate that the crab defense system was overwhelmed. It is important to note that this condition was evident at the end of the experiment (day 7), where (1) oxygen consumption was higher, (2) CAT activity was lower, and (3) activities of antioxidant enzymes (GST and SOD) were not different from those in the control group. Ding et al. (1998a) and Guzman and Solter (1999) also showed augmented levels of LPO in rat hepatocytes after microcystin exposure.
Hepatopancreatic B cells are involved in intracellular digestion and express P-glycoprotein (P-gp) (Al-Mohanna and Nott, 1989; Ko¨hler et al., 1998). According to Ko¨hler et al. (1998), the P-gp system is involved in the transport of toxins from the cytoplasm to the lysosomal compartment for later elimination by senescence of B cells, being expelled into the digestive lumen. The fact that crabs exposed to microcystin showed a low number of B cells suggests an involvement of P-gp in the defensive mechanism of crab hepatopancreas against microcystins, leading to a decrement in B cell number. As verified in different studies, microcystin is highly toxic to several animal species, with values of LD50 varying approximately between 50 and 550 mg/kg (Tencalla et al., 1994; Yunes et al., 1996; Matthiensen et al., 2000). However, few studies have analyzed the in vivo biochemical responses after administration of sublethal doses of microcystin. For example, Guzman and Solter (1999) exposed rats during 28 days to doses of 16, 32, and 48 mg/kg/day of microcystin, verifying oxidative damage with respect to augmented LPO levels and histopatologies. In the present study, microcystin effects such as oxidative stress and antioxidant responses were observed when employing doses as low as 1.06 and 5.32 mg/kg/day and in a shorter period of time (7 days). Based on data reported by Vasconcelos et al. (2001), it can be estimated that the crayfish Procambarus clarkii accumulated approximately 0.73 mg of microcystin/kg of wet weight. In another study (Vasconcelos, 1995), it was determined that microcystin accumulation in the mussel Mytilus galloprovincialis was about 2.63 mg of microcystin/kg of wet weight. These two values are extremely high compared with the estimated total amount of microcystin administered to C. granulatus in the present study. In the present study, crabs were forced to ingest 14.31 or 71.54 mg of microcystin per day. Thus, the total amount of toxin administered to the crabs were 7.5 and 37.2 mg/kg of wet weight, respectively. These values are close to those determined by Magalha˜es et al. (2001) in muscle, liver, and viscera of the fish Tilapia randelli in areas where blooms of Microcystis sp. have occurred. Overall, these results suggest that toxic effects associated with oxidative damage must be determined simultaneously with determinations of toxin accumulation, if the objective is to evaluate deleterious effects of microcystin to aquatic fauna. In conclusion, the present results indicate that microcystin increases oxygen consumption, modifies enzymatic and nonenzymatic antioxidant defenses (CAT, GST, and NP-SH content), and induces oxidative damage with regard to LPO in different crab tissues. These results also indicate that oxidative stress is involved in toxicity of microcystin. Further studies should consider mechanisms of cellular absorption and elimination of microcystin to generate a better
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understanding of its mechanism of toxicity through oxidative stress generation in invertebrates.
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