Introduction
All aerobic organisms are exposed to a wide variety of environmental disturbance. Heat stress is one of the most important external stresses for survival, and it has been found to increase oxidative damage by accelerating the generation of reactive oxygen species (ROS). Although small amounts of ROS are a cellular requirement because they are involved in signalling pathways, regulating a variety of cellular activities and gene expression, excess ROS production increases oxidative damage in the cell, possibly by altering or inactivating proteins, lipid membranes, and DNA (Hensley et al. Reference Hensley, Robinson, Gabbita, Salsman and Floyd2000). Although our previous work found that thermal stress on the Antarctic stenothermal clam Laternula elliptica elevated expression of the Hsp70 gene (Park et al. Reference Park, Ahn and Lee2007), understanding of the mechanism about oxidative stress from heat shock is still lacking. Heise et al. (Reference Heise, Puntarulo, Portner and Abele2003) found that ROS formation in mitochondrial isolates from L. elliptica is significantly elevated under temperature stress, and also that ROS formation is positively correlated with oxygen consumption. ROS generation by both a temperature increase and enhanced oxygen consumption may promote oxidation of cellular constituents and a response by antioxidant and associated enzyme systems.
Antioxidants help prevent cellular damage caused by metabolically and environmentally produced ROS, such as hydrogen peroxide (H2O2) and superoxide anion radicals (O2-). Organisms can respond to a high ROS concentration by increasing the expression of the antioxidant system, including enzymatic (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; glutathione S-transferase, GST; glutathione reductase, GRd) and nonenzymatic components such as cellular thiol compounds. GRd regenerates reduced glutathione (GSH), which is a direct scavenger of ROS or a substrate for GPx (Halliwell & Gutteridge Reference Halliwell and Gutteridge1999). GST also plays an important role in the detoxification of ROS in cells (Edwards et al. Reference Edwards, Dixon and Walbot2000) by protecting lipids from peroxidation. Cellular thiol status is also of great importance in the antioxidant defence system. The tripeptide GSH (γ-glutamyl-cysteinyl-glycine) is one of the most important antioxidant agents, which participates in reducing buffers that maintain the reduced/oxidized ratio (Meister & Anderson Reference Meister and Anderson1983, Schafer & Buettner Reference Schafer and Buettner2001). Additionally, protein modifications caused by direct oxidative attack on Lys, Arg, Pro, or Thr residues by reactive carbonyl compounds can lead to the formation of protein carbonyl (PC) derivatives (Stadtman & Levine Reference Stadtman and Levine2000). Hence, PCs are considered a broad marker of oxidation, as elevated PC levels are generally signs of oxidative stress.
Previous work on the role of antioxidant defences in thermal stress using L. elliptica at the cellular level has reported only the responses of two mitochondrial antioxidant enzymes and lipid peroxidation (Estevez et al. Reference Estevez, Abele and Puntaruloa2002, Heise et al. Reference Heise, Puntarulo, Portner and Abele2003). Studies of other antioxidant enzymes and antioxidant components are still lacking. The objective of this study, therefore, was to investigate the change in activity of several oxidative enzymes, including SOD, CAT, GPx, GRD, and GST, in digestive gland and gill tissues of L. elliptica when exposed to thermal stress. We also include the GSH and PC contents as markers of oxidative stress.
Materials and methods
Animal collection and heat exposure
Laternula elliptica (shell length ~80 mm) were hand-collected by SCUBA divers at depths of 20–30 m in Marian Cove, near King Sejong Station, King George Island (62°13′S, 58°47′W) in January 2006. The samples were divided into two groups and each was acclimated under conditions equivalent to those in the field at a temperature of c. 1.0°C with constant air supply. After acclimation for two days, one group was subjected to 10 ± 1°C as acute heat stress, which is thermal limit for survival (Peck et al. Reference Peck, Portner and Hardewig2002), and the other group kept as a control (1.0 ± 1°C) with well-aerated natural sea water and without feeding.
Tissue preparation and enzyme assays
The clams from each group were killed, and digestive gland and gill tissue were dissected immediately after their exposure. The tissues were homogenized in buffer (50 mmol L-1 phosphate buffered saline (pH 7.4) and 1 mmol L-1 EDTA) containing an antiprotease cocktail (10 mg ml-1 antipain, pepstatin A, leupeptin, and soybean trypsin inhibitor, plus 2.5 mmol L-1 PMSF). Antioxidant enzymes were assayed at 2°C, similar to the temperature of the clam's habitat in the Southern Ocean, using a thermostatic spectrophotometer (HP8453; Hewlett-Packard, Palo Alto, CA, USA). SOD activity was assayed by the xanthine oxidase/cytochrome c method (McCord & Fridovich Reference McCord and Fridovich1969). The reaction mixtures contained 10 uM cytochrome c and 50 uM xanthine in 50 mM sodium carbonate, 0.1 mM EDTA, at pH 10. Reactions were started by adding xanthine oxidase in an amount sufficient to cause A 550 nm of 0.025/min in the cytochrome c assay. One unit of SOD was defined as the amount of sample needed to achieve 50% inhibition. CAT activity was assayed by the method of Beers & Sizer (Reference Beers and Sizer1952). The decomposition of H2O2 (ε = 0.0394 mmol L-1 cm-1) was followed directly by measuring the decrease in absorbance at 240 nm. One unit of CAT was defined as the amount of enzyme decomposing 1 umol of H2O2 in 1 min at pH 7.0, and specific activity was given as U per mg protein. GPx activity was measured in a coupled enzyme system in which the oxidized GSH formed in the GPx reaction was converted to the reduced form GSH by GRd. The consumption of NADPH was monitored at 340 nm and was directly proportional to the GPx activity in the sample, which was expressed as U per mg protein. GRd activity was determined according to the method of Carlberg & Mannervik (Reference Carlberg and Mannervik1985), measuring the oxidation of NADPH to NADP+ by GRd in the presence of oxidized glutathione. The reduction of GSSG was determined indirectly by measuring the oxidation rate of NADPH, as visualized by the time-dependent decrease in absorbance at 340 nm. One GRd unit was defined as the reduction of 1 µmol of GSSG min-1 at pH 7.6, and specific activity was expressed as U per mg protein. GST activity was measured according to the method of Habig et al. (Reference Habig, Pabst and Jakoby1974) using 1-chloro-2,4-dinitbenzene (CDNB) and GSH as substrates. A unit of GST was defined as the amount of glutathione conjugate formed using 1 mM GSH and CDNB min-1 mg protein. All data was expressed as the mean ± SD and analysed by Student's t-test. Differences were considered significant at P < 0.05.
Glutathione content and protein carbonyl content assays
Tissues were homogenized in 5 vol of cold 1 M perchloric acid containing 2 mM EDTA and centrifuged at 30 000 g for 20 min. An aliquot of the pellet was used for PC content measurement. The supernatant was neutralized with 2 M KOH/0.3 M MOPS and centrifuged at 10 000 g for 10 min at 4°C. The total glutathione concentration was evaluated in the neutralized supernatant by the glutathione reductase enzymatic assay (Akerboom & Sies Reference Akerboom and Sies1981). The reaction mixture contained 0.1 M potassium-phosphate buffer, 1 mM EDTA (pH 7), 0.2 mM NADPH, 0.06 mM DTNB, and 0.12 units glutathione reductase. The linear increase in absorbance at 412 nm and 25°C was recorded with a spectrophotometer (HP8453). A blank assay, without GSH, was run separately. For calibration, the procedure was performed using 100 uM GSSG instead of sample.
PC derivatives were detected by reaction with 2,4-dinitrophenylhydrazine (DNPH). This compound reacts with PC groups that have been formed by ROS attack on side chains of amino acids. Resulting 2,4-dinitrophenylhydrazones were quantified spectrophotometrically at 370 nm using a molar extinction coefficient of 22 × 103 M-1 cm-1 (Lenz et al. Reference Lenz, Costabel, Shaltiel and Levine1989). The pellet from the TCA extract (above) was mixed with 1 ml of 10 mM DNPH in 2 M HCl. Control samples contained 1 ml of 2 M HCl instead the DNPH solution. Samples were incubated for 1 h at room temperature, and then centrifuged for 10 min at 5000 g. Supernatants were discarded and pellets were washed three times with 1 ml of ethanol–butylacetate (1:1 v/v) mixture, and pellets were dissolved in 1.5 ml of 6 M guanidine-HCl. The values were expressed as nanomoles of PC per gram wet weight of tissue. All data was expressed as the means ± SD and analysed by Student's t-test. Differences were considered significant at P < 0.05.
Results
Figures 1a & 2a show the effects of thermal stress on SOD activity in digestive gland and gill tissues of L. elliptica. After 12 h of exposure to heat stress, SOD activity increased slightly; it peaked after one day in both digestive gland and gill tissues. After one day, SOD activity decreased gradually and returned to the control level in both organs. Another primary antioxidant enzyme, CAT, was not significantly affected by experimental condition (Figs 1b & 2b).
Antioxidant enzyme activities in digestive gland tissues of control (circles and line) and thermal-exposed (bars) L. elliptica. a. SOD, b. CAT, c. GPx, d. GRd, e. GST. Data are mean ± SD, n = 3–5. Asterisks indicate significant differences (P ≤ 0.05) from the control treatment. Enzymatic activity is expressed in specific units (U) as defined in the materials and methods section.
Antioxidant enzyme activities in gill tissues of control (circle and line) and thermal-exposed (bar) L. elliptica. a. SOD, b. CAT, c. GPx, d. GRd, e. GST. Data are mean ± SD, n = 3–5. Asterisks indicate significant differences (P ≤ 0.05) from the control treatment. Enzymatic activity is expressed in specific units (U) as defined in the materials and methods section.
The activities of GSH-related antioxidant enzymes were strongly affected by exposure to thermal stress. GPx activity increased in digestive gland (about -1.7 fold) and gill (about -1.8 fold) after two days of heat exposure, although it remained constant for the first 12 h of treatment (Figs 1c & 2c). The activity of GRd was significantly altered by 1.9-fold in digestive gland and 2.1-fold in gill at the two day exposures (Figs 1d & 2d). Compare to the control, GST activity was the most sensitive to heat stress, increasing more than 3-fold and 4-fold in digestive gland and gill, respectively (Figs 1e & 2e).
The concentration of total GSH in L. elliptica tissues continuously increased under heat stress. After two days of heat treatment, GSH levels had increased by 1.9- and 4.4-fold in digestive gland and gill tissues, respectively (Fig. 3a). Heat stress had little effect on PC content on the first day of heat exposure, but after two days, PC levels increased slightly (Fig. 3b).
Effect of heat exposure on a. GSH level, and b. PC content in digestive gland (filled bars) and gill (empty bars) tissues of L. elliptica. Data are mean ± SD, n = 3–5. Asterisks indicate significant differences (p ≤ 0.05) from the control treatment.
Discussion
An increase in environmental temperature can lead to increased oxygen consumption, which in turn promotes ROS generation (Abele et al. Reference Abele, Burlando, Viarengo and Pörtner1998, Reference Abele, Tesch, Wencke and Pörtner2001). Heise et al. (Reference Heise, Puntarulo, Portner and Abele2003) reported the thermal stress induced significantly elevated ROS formation and significantly higher lipid radical generation in L. elliptica than in a temperate mud clam.
SOD activity, as it catalyses the removal of oxygen radical metabolically derived oxidative stress, has been shown to respond to thermal stress in digestive gland and gill tissues of L. elliptica, although the level of CAT activity increased slightly but not significantly in response to thermal stress. CAT directly reduces H2O2, which is produced by SOD, to produce water and oxygen. These two antioxidant enzymes are distributed differently in digestive gland and gill. SOD activity was much higher in gill than in digestive gland, whereas CAT and other antioxidant enzymes were more active in digestive gland than in gill. Whereas Viarengo et al. (Reference Viarengo, Canesi, Garcia, Peters and Livingstonet1995) found higher SOD activity in digestive gland than in gill of the Antarctic mollusc Adamussium colbecki, Regoli et al. (Reference Regoli, Principato, Bertoli, Nigro and Orlando1997) found that SOD activity is significantly higher in gill than in digestive gland. These contrasts with different enzyme activities between gill and digestive gland make it difficult to distinguish whether the antioxidant defences can be simultaneously or differentially modulated by stress in polar molluscs.
This is the first report on the relationship between GSH-related antioxidant enzymes and thermal stress in Antarctic stenothermal bivalves. Antioxidant enzymes connected to GSH metabolism showed significant responses to thermal stress in clam tissues. GPx activity increased over 1.7-fold in both tissues after heat exposure. Whereas CAT directly metabolizes H2O2 to ground-state O2 and water, GPx, the major degradative enzyme for H2O2 and lipid hydroperoxides, couples the reduction of H2O2 to water with oxidation of reduced GSH. GSH, a ubiquitous cellular nonprotein sulphydryl compound, is involved in several cellular functions (Meister & Anderson Reference Meister and Anderson1983, Reed Reference Reed1990). As a hydrogen donor and radical scavenger, GSH plays a major role in processes inducing or generating free radicals (Carmel-Harel & Storz Reference Carmel-Harel and Storz2000). It is not clear whether a certain level of GSH is necessary to develop thermal tolerance; intracellular concentrations of GSH increase after exposure to heat shock in several cells (Mitchell et al. Reference Mitchell, Russo, Kinsella and Glatstein1983, Konings & Penninga Reference Konings and Penninga1985, Skibba et al. Reference Skibba, Stadnicka, Kalbfleisch and Powers1989, Harris et al. Reference Harris, Juchau and Mirkes1991), and it has been suggested that GSH is needed to mediate the initial response necessary for acquiring heat stress tolerance (Russo et al. Reference Russo, Mitchell and McPherson1984). Although few studies have reported on the influence of heat stress on GSH metabolism, these data suggest that the GSH redox system via GPx is robustly maintained in the face of heat-induced oxidative stress. GSH disulphide is reduced by GRd to form GSH; GRd activity is essential in the maintenance of GSH in its reduced form (Pena-Llopis et al. Reference Pena-Llopis, Ferrando and Pena2003). The activity of GRd was also significantly altered by heat stress. Heat stress appears to both produce oxidants and impair the enzyme system necessary for detoxification of H2O2 and organic hydroperoxides. Another GSH-related enzyme, GST, plays an important role in the detoxification of foreign compounds (Ceballos-Picot et al. Reference Ceballos-Picot, Nicole, Clement, Bourre and Sinet1992). In this study, GST activity was the most sensitive to heat stress. These increases in activity might have resulted from the increased lipophilic substances released during oxidative damage to polyunsaturated lipids, such as short-chain aldehydes and alkenals, which are substrates for GST (Galli et al. Reference Galli, Rovidati, Benedetti, Buoncristiani, Covarelli, Floridi and Canestrari1999). Peck et al. (Reference Peck, Portner and Hardewig2002) reported that 50% mortality was observed within seven days exposure at 9°C because L. elliptica has low upper temperature limits and it transfers to anaerobic metabolism in this temperature condition, whereas L. elliptica mortality was not observed during the experiments in this study. Pörtner et al. (Reference Pörtner, Peck and Hirse2006) suggested that survival, at this extreme temperature, is supported by anaerobic metabolism and the protection of protein and membrane function by heat shock proteins and antioxidative defence. Previous studies have already shown that transcription level of heat shock protein to protein protection was instantly increased for thermal stress (Park et al. Reference Park, Ahn and Lee2007). Our findings in this study indicate that phase II detoxification enzyme system was an important protection function at oxygen-limited thermal stress, and it might be involved in metabolism of accumulated anaerobic end products (Peck et al. Reference Peck, Portner and Hardewig2002).
The levels of total GSH concentration were affected by heat stress. GSH may contribute to protection from ROS, but, at the same time, it is involved in the regulation of many redox-sensitive processes (Droge Reference Droge2002). Under oxidative stress, GSH content often rises due to de novo synthesis (Ali et al. Reference Ali, Parvez, Pandey, Atif, Kaur, Rehman and Raisuddin2004, Lushchak et al. Reference Lushchak, Bagnyukova, Lushchak, Storey and Storey2005). In our results, GSH increased continuously during thermal stress and changed in a similar manner to GSH-related antioxidant enzymes. We suggest that cellular GSH is involved in the major defence against thermal-induced oxidative stress. By contrast, PC contents in both organs slightly increased after two days of heat exposure. Compared to other oxidative modifications, PC are relatively difficult to induce and are irreversible modifications (Dalle-Donne et al. Reference Dalle-Donne, Giustarini, Colombo, Rossi and Milzani2003). The level of carbonylated proteins is closely linked to ROS generation, so it is probable that increases in PC level might be shown as a results of irreversible physiological damage after exhausting the protective potential such as the action of chaperones.
Conclusions
This study represents the first comprehensive report of antioxidant defence enzyme activities and antioxidant components in the cold stenothermal L. elliptica from the Southern Ocean. Our results indicate that the GSH-related enzymes GRd, GPx, and GST are likely to be important in the antioxidant defence system at heat shock exposure, whereas SOD and CAT play a minor role in thermal stress. GSH metabolism is also greatly involved in the antioxidant defence system in L. elliptica under oxidative stress induced by thermal stress but protein oxidation was not as PC levels were not affected instantly by heat shock. Our present analysis is limited by extreme thermal stress, so further investigation will be needed to study antioxidative defences dependence on temperature variation under such critical temperatures, and with aerobic metabolism. Such integrative approaches to antioxidant defence system ins L. elliptica subjected to thermal stress contributes to our knowledge of adaptation in extremely cold environments.
Acknowledgements
This study was supported by Monitoring on Environmental Changes at the Korean Arctic and Antarctic stations (PE08040), funded by Korea Polar Research Institute (KOPRI) in the Korea Ocean Research & Development Institute (KORDI).
