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Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney
Comparative Biochemistry and Physiology, Part B 142 (2005) 335 – 341 www.elsevier.com/locate/cbpb
Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney Tetyana V. Bagnyukova a, Kenneth B. Storey b, Volodymyr I. Lushchak a,* a
Department of Biochemistry, Precarpathian National University named after Vassyl Stefanyk, 57 Shevchenko Str., 76025, Ivano-Frankivsk, Ukraine b Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6 Received 7 April 2005; received in revised form 17 August 2005; accepted 18 August 2005 Available online 15 September 2005
Abstract This study was undertaken to clarify the physiological role of catalase in the maintenance of pro/antioxidant balance in goldfish tissues by inhibiting the enzyme in vivo with 3-amino 1,2,4-triazole. Intraperitoneal injection of aminotriazole (0.5 mg/g wet mass) caused a decrease in liver catalase activity by 83% after 24 h that was sustained after 168 h post-injection. In kidney catalase activity was reduced by ¨50% and 70% at the two time points, respectively. Levels of protein carbonyls were unchanged in liver but rose by 2-fold in kidney after 168 h. Levels of thiobarbituric acid-reactive substances were elevated in both tissues after 24 h but were reversed by 168 h. Glutathione peroxidase and glutathione-S-transferase activities increased in kidney after aminotriazole treatment whereas activities of glutathione peroxidase and glutathione reductase in liver decreased after 24 h but rebounded by 168 h. Liver glucose-6-phosphate dehydrogenase activity was reduced at both time points. Activities of these three enzymes in liver correlated inversely with the levels of lipid damage products (R 2 = 0.65 – 0.81) suggesting that they may have been oxidatively inactivated. Glutathione-S-transferase activity also correlated inversely with catalase (R 2 = 0.86). Hence, the response to catalase depletion involves compensatory changes in the activities of enzymes of glutathione metabolism. D 2005 Elsevier Inc. All rights reserved. Keywords: 3-Amino 1,2,4-triazole; Carassius auratus; Carbonyl proteins; Glutathione peroxidase; Glutathione-S-transferase; Kidney; Liver; Thiobarbituric reactive substances
1. Introduction All aerobic organisms need molecular oxygen for their oxidative metabolic processes but as a consequence must also deal with the formation of dangerous reactive oxygen species (ROS) including superoxide anion (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (UOH) (Halliwell and Gutteridge, 1989). ROS attack and chemically alter cellular macromolecules including proteins, lipids and DNA causing metabolic damage that is often severe. Therefore, all organisms possess well-developed systems of antioxidant defense that include both low-molecular weight antioxidants and antioxidant enzymes. The main antioxidant enzymes are superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Under normal physiological conditions there is the balance between ROS generation and their elimination by different * Corresponding author. Fax: +1 38 03422 31574. E-mail address: [email protected] (V.I. Lushchak). 1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2005.08.003
antioxidant scavengers. If prooxidant processes are enhanced and/or the power of the antioxidant system decreased, oxidative stress arises (Sies, 1991). Although all protective components are needed to cope with oxidative stress, each particular enzyme accomplishes specific functions that can only be partly replaced by the actions of other antioxidant defenses. There are two enzymes that catabolize hydrogen peroxide. Catalase converts H2O2 to water and molecular oxygen, thus preventing the formation of extremely dangerous hydroxyl radical from H2O2 via the Fenton reaction (Kehrer, 2000). GPx degrades H2O2 using reduced glutathione. The cooperation between catalase and GPx in dealing with cellular H2O2 has been the subject of a number of investigations. In some studies, catalase seemed to be more important than GPx in detoxifying H2O2 (Barja de Quiroga et al., 1988, 1989; Bouzyk et al., 1997). Other studies, however, reported that GPx was more powerful in antioxidant defense (Hiraishi et al., 1991; Remacle et al., 1992; Dorval and Hontela, 2003). To elucidate the
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significance of an enzyme, specific inhibitors are widely used. For example, catalase can be irreversibly inhibited by 3-amino 1,2,4-triazole (AMT) (Margoliash et al., 1960). The goldfish, Carassius auratus L., is well adapted to varied oxygen concentrations, from hyperoxia to hypoxia and even anoxia (van den Thillart and van Waarde, 1985). This fish possesses potent antioxidant defenses, as confirmed by high antioxidant enzyme activities as well as glutathione levels, particularly in two metabolically active tissues, liver and kidney (Lushchak et al., 2001). These tissues are sites of xenobiotic detoxification and, therefore, are considered to be powerful ROS generators (Halliwell and Gutteridge, 1989). The aim of this study was to investigate the effect of catalase inhibition by AMT on markers of oxidative stress and the activities of selected antioxidant enzymes in liver and kidney in order to elucidate possible mechanisms that could compensate for catalase function when this enzyme was inhibited. 2. Materials and methods 2.1. Chemicals Phenylmethylsulfonyl fluoride (PMSF), butylated hydroxytoluene (BHT), yeast glutathione reductase (GR), 1-chloro-2,4dinitrobenzene (CDNB), reduced glutathione (GSH), oxidized glutathione (GSSG), glucose-6-phosphate (G6P), ethylenediamine-tetraacetic acid (EDTA), sodium azide, and 3-amino 1,2,4-triazole (AMT) were purchased from Sigma Chemical Co. (USA). N,N,NV,NV-tetramethylethylenediamine (TEMED), NADP+ and NADPH were from Reanal (Hungary), Tris – HCl was from BioRad, and guanidine – HCl from Fluka. All other reagents were of analytical grade. 2.2. Animals and experimental conditions Goldfish (C. auratusL.) of both sexes weighing 20 – 70 g were purchased at a local fish market (Ivano-Frankivsk, Ukraine) and were kept in dechlorinated tap water and fed with standard goldfish food. Temperature was maintained at 18 T 1 -C with a natural light– dark cycle with light from about 8:00 to 17:00 h. Goldfish were acclimated to these conditions for at least one month before experimentation. For preliminary investigation of the effect of AMT on catalase inhibition, fish were injected intraperitoneally with AMT diluted in physiological saline at final concentrations of 0.1, 0.5 or 1.0 mg/gram wet body mass (gww). The volume of injected solution was 0.45% of body weight. Sham-injected fish received 0.9% NaCl solution instead AMT solution. Fish were killed by transspinal dissection at 5, 10, 24, 48, 72, 120 and 168 h after the treatment and catalase activity was measured immediately in liver and kidney. For subsequent studies, fish were injected with AMT solution at a final concentration of 0.5 mg/gww (the volume was 0.3% of body mass). A sham-injected group was treated with the same volume of 0.9% NaCl. Control fish were not treated. After 24 or 168 h the fish injected with AMT or NaCl solutions were sampled for organ dissection. For sampling, fish were killed and
the liver and kidneys were quickly removed. The samples were immediately used to measure the parameters of interest. 2.3. Indices of oxidative stress Tissue samples were homogenized (1 : 10 w/v) using a Potter –Elvjeham glass homogenizer in 50 mM potassium phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA and a few crystals of PMSF, a protease inhibitor. A 250 AL aliquot of this homogenate was then mixed with 0.5 mL of 10% (final concentration) trichloroacetic acid (TCA) and centrifuged for 5 min at 13,000 g in a microcentrifuge. Levels of protein carbonyls (CP) were measured in the resulting pellets, and thiobarbituric acid reactive substances (TBARS) contents were assayed in the supernatants using a spectrophotometer SF-46 (LOMO, Russia). Carbonyl derivatives of proteins were detected by reaction with 2,4-dinitrophenylhydrazine (DNPH). Resulting 2,4-dinitrophenylhydrazones were quantified spectrophotometrically (Lenz et al., 1989) with minor changes to the assay procedure (Bagnyukova et al., 2003). Briefly, the pellets from TCA treated extracts (above) were incubated with 1 mL of 10 mM DNPH in 2 M HCl (samples) or 1 mL of 2 M HCl only (controls). After incubation, samples were centrifuged, and resulting pellets were washed three times with ethanol – ethylacetate (1 : 1, v/v). Pellets were then dissolved in 6 M guanidine – HCl. The amount of CP was evaluated spectrophotometrically at 370 nm using a molar extinction coefficient of 22I103 M 1 cm 1 (Lenz et al., 1989). The values were expressed as nanomoles of CP per milligram protein in the guanidine chloride solution. The decomposition of lipid hydroperoxides produces lowmolecular weight products, including malondialdehyde, which can be measured by the TBARS assay (Rice-Evans et al., 1991). The supernatants from the TCA extract (above) were combined with the same volume of TBA reagent, containing a saturated solution of TBA in 0.1 M HCl and 10 mM BHT previously dissolved in ethanol; pH was adjusted to 2.5. BHT was added to avoid tissue peroxidation during heating of the samples. Control samples contained water instead of supernatant. The mixtures were immersed in a boiling water bath for 60 min. After quick cooling, a volume of butanol equal to the mixture total volume was added and mixed vigorously. Samples were centrifuged for 10 min at 5000 g and the butanol phase was removed and used to evaluate the level of TBARS. Absorption was measured at 535 nm and a molar extinction coefficient of 156 103 M 1 cm 1 was used to calculate TBARS concentration (Rice-Evans et al., 1991). The values are expressed as nanomoles of TBARS per gram wet weight (gww) of tissue. 2.4. Assay of antioxidant enzyme activities Tissue homogenates prepared as for the TBARS/CP assay were centrifuged at 4 -C for 15 min at 15,000 g. Supernatants were removed and used for enzyme activity assays using a spectrophotometer SF-46 (LOMO, Russia). The activity of superoxide dismutase (SOD) was assayed as a function of its inhibitory action on quercetin oxidation
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(Bagnyukova et al., 2003). The reaction mixture contained (final concentrations): 30 mM Tris – HCl buffer (pH 10.0), 0.5 mM EDTA, 0.8 mM TEMED, 0.05 mM quercetin, and 1.5 –50 AL of supernatant. The reaction was monitored at 406 nm for 6 – 8 different volumes of supernatant. One unit of SOD activity is defined as the amount of enzyme (per milligram protein) that inhibits the quercetin oxidation reaction by 50% of maximal inhibition. In our case, the maximal inhibition was about 90%. Catalase activity was measured at 240 nm in a medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 10 mM H2O2, and 2– 100 AL of supernatant (Bagnyukova et al., 2003). Blanks were run without hydrogen peroxide and/or supernatant. Selenium-dependent glutathione peroxidase (GPx) activity was measured by a coupled assay linked with GR catalyzed oxidation of NADPH (Lushchak et al., 2001). A basal consumption of NADPH was recorded at 340 nm in a medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 0.25 mM NADPH, 4 mM sodium azide, 1 U/mL GR, 15 mM GSH, and 2 –50 AL of the supernatant. Then, 10 AL of H2O2 was added to a final concentration of 0.2 mM (final volume, 1 mL). Blanks contained no supernatant. Glutathione-S-transferase (GST) activity was measured by monitoring the formation of an adduct between GSH and CDNB at 340 nm (Lushchak et al., 2001). Reaction mixture contained 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 5 mM GSH, 1 mM CDNB and 2 – 7 AL of supernatant in a final volume of 1 mL. The reaction was initiated by the sequential addition of CDNB and supernatant. Blanks contained no CDNB. The activity of GR was assayed at 340 nm in medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 0.25 mM NADPH, 1 mM GSSG, and 40– 50 AL of supernatant (Bagnyukova et al., 2003). Blanks omitted GSSG. Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured at 340 nm in 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 5 mM MgCl2, 0.2 mM NADP, and 2 mM G6P with 30 – 50 AL of supernatant (Bagnyukova et al., 2003). Blanks were run without G6P. One unit of catalase, GPx, GST, GR, or G6PDH activity is defined as the amount of the enzyme consuming 1 Amol of substrate or generating 1 Amol of product/ min; activities were expressed as international units (or milliunits) per milligram protein. 2.5. Protein measurements and statistics Protein concentration was measured by the Bradford method with Coomassie Brilliant Blue G-250 (Bradford, 1976) and using bovine serum albumin as a standard. Data are presented as means T SEM. Statistical analysis was performed using Student’s t-test. Inhibition values for SOD activity were calculated using an enzyme kinetics computer program (Brooks, 1992). Correlation analysis was performed using Excel software. 3. Results and discussion Goldfish liver and kidney have well-developed antioxidant systems that include both antioxidant enzymes and lowmolecular weight antioxidants such as glutathione. Goldfish
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liver shows particularly well-developed defenses with activities of catalase, GPx and GST that were 3- to 5-fold higher than in kidney whereas SOD, GR and G6PDH activities were similar in both tissues (Table 1). These values were similar to (catalase, GST) or lower than (GR, G6PDH) enzyme activities obtained in an earlier study that used vual tail goldfish (Lushchak et al., 2001). That study also showed that total glutathione content was 3.3-fold higher in goldfish liver than in kidney (Lushchak et al., 2001). It is known that AMT irreversibly inactivates catalase (Margoliash et al., 1960). A single AMT injection resulted in a quick decrease in catalase activity in frog liver and kidney and activity remained reduced over at least 6 days (Barja de Quiroga et al., 1989; Lushchak et al., 2003). In a preliminary study we administered AMT to goldfish at 0.1, 0.5 or 1.0 mg/ gww and measured its effects on catalase activity in liver and kidney at times ranging from 5 to 168 h after injection. AMT injection affected catalase activity in both dose- and timedependent manners (data not shown). Catalase activity decreased by more than 60% in liver within 5 h after injection with AMT (as compared with sham-injected animals) and remained at this level over the remainder of the experimental course. A similar result was found in kidney with the major decrease in enzyme activity by 50– 90% seen within the first 24 h of treatment; subsequently, activities remained low over the rest of the time course. Using these preliminary data, we chose treatment with AMT at a concentration of 0.5 mg/gww for 24 or 168 h as the design for a second set of experiments. Fig. 1 shows that injection with AMT at a dose of 0.5 mg/ gww caused a sharp decrease in catalase activity in liver. Activity had dropped to ¨17% of the value in control (uninjected) fish after 24 h and remained at this level after 168 h post-injection. In kidney, catalase activity was reduced by 50% after 24 h post-injection and by 66% after 168 h of treatment. Thus, a single AMT injection was enough to cause a strong decrease in organ catalase activities that lasted for at least 7 days. Sham injection of fish with physiological saline alone did not affect catalase activity in either liver or kidney; activities were not significantly different from control values at either 24 or 168 h post-injection. Experimental treatments caused some alterations in organ protein concentrations. Liver of fish sampled 168 h after injection showed a 50% increase in soluble protein content for both sham-injected and AMT-injected fish (Table 2). By contrast, protein content was slightly reduced in kidney after AMT treatment; a significant decrease of 16% was seen after 24 h and the level remained low at 168 h. Although we do not Table 1 Activities of antioxidant enzymes in liver and kidney of goldfish (n = 5 – 9) Enzyme
Liver
Kidney
SOD, U/mg Catalase, U/mg GPx, mU/mg GST, U/mg GR, mU/mg G6PDH, mU/mg
74.7 T 17.9 58.5 T 12.7 533 T 61 1.45 T 0.17 8.17 T 0.69 44.0 T 7.6
50.1 T16.9 13.1 T1.3 109 T 7 0.46 T 0.06 11.4 T 1.4 39.0 T 4.4
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16
1,4
CP, nmol/mg protein
60
40
20
0
1,2
1
2
3
1,4
4
5
Liver
1,2
1 2
3
12 1
8
4
1,4 4
0
5
1
2
Kidney
3
4
5
Liver
Fig. 1. The effect of AMT on catalase activity in goldfish liver and kidney. Experimental conditions are: (1) control uninjected fish, (2) 24 h post-injection with 0.9% NaCl, (3) 24 h post-injection with 0.5 mg/gww AMT, (4) 168 h postinjection with 0.9% NaCl (5) 168 h post-injection with 0.5 mg/gww AMT. Data are means T SEM, n = 5 – 7 fish. Numbers over the bars indicate significant differences from the indicated groups, P < 0.05.
know which types of proteins were affected, these changes may be considered as a general response to stress caused by saline or AMT. Catalase inhibition resulted in the development of oxidative stress in goldfish tissues which was confirmed by elevated levels of CP and TBARS in some cases. The amount of oxidatively damaged proteins, measured by CP levels, did not change in liver under any experimental condition but in kidney of AMT-treated fish CP content rose 2.4-fold after 168 h (Fig. 2). Levels of CP in kidney were also slightly elevated in shaminjected fish sampled at 168 h. Both tissues showed a similar pattern of change in TBARS levels, reflecting oxidative damage to lipids. TBARS content rose by 2- and 3-fold in liver and kidney, respectively, after 24 h post-injection but levels returned to control values by 168 h (Fig. 3). The different patterns of change in CP and TBARS levels probably reflect different cellular responses to oxidative damage to proteins versus lipids. Several explanations for this may be proposed. Firstly, cell proteins and lipids may have different susceptibilities to oxidation by ROS. Secondly, different defenses are available in cells to deal with oxidative damage to lipids and proteins. Lipid peroxides are decomposed by phospholipid Table 2 Soluble protein concentration (mg/mL) in liver and kidney of goldfish Groups
Liver
Kidney
Control 24 h NaCl 24 h AMT 168 h NaCl 168 h AMT
5.20 T 0.51 3.74 T 0.72 6.35 T 0.38* 7.97 T 0.74C 7.81 T 0.79C
4.19 T 0.27 4.07 T 0.40 3.53 T 0.22C 4.27 T 0.35 3.59 T 0.27
NaCl and AMT groups were injected with physiological saline or AMT, respectively, and sampled after 24 or 168 h. Protein was measured in the supernatants prepared for enzyme assay. C Significantly different from the corresponding control value or *from the group treated for 24 h with NaCl ( P < 0.05), n = 6 – 9.
1
2
3
4
5
Kidney
Fig. 2. The effect of AMT on protein carbonyl levels in goldfish liver and kidney. Other information as in Fig. 1.
hydroperoxide glutathione peroxidase (Hermes-Lima, 2004) whereas oxidized proteins are degraded by specific and nonspecific proteases whose activities depend, in turn, on a variety of factors and may be inhibited by ROS and their products (Stadtman and Levine, 2000). Thirdly, long-term differences in the accumulation and tolerance of different types of damaged macromolecules occur; for example, during aging and under prolonged stress conditions oxidized proteins tend to be accumulated (Stadtman and Levine, 2000). It appears that the inherent antioxidant defenses of goldfish liver were still able to protect hepatic proteins from oxidation damage even when catalase was depleted due to inhibition by AMT, a situation where intracellular H2O2 levels would presumably be very high. The greatly reduced activity of catalase might be compensated for in other ways, for example, by glutathione-linked enzymes. The same is true to a lesser extent for kidney where significant CP accumulation was seen only after 168 h. With respect to TBARS content, liver and kidney showed an early sharp increase, by two- to three-fold, 80 1,2
TBARS, nmol/gww
Catalase, U/mg protein
80
60
1,2
1
40
20
0
1
2
3
Liver
4
5
1 2
3
4
5
Kidney
Fig. 3. The effect of AMT on the levels of thiobarbituric acid-reactive substances in goldfish liver and kidney. Other information as in Fig. 1.
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600
400 1,4
1,2 1
200
0 1
2
3
4
Liver
5
1
2
3
4
5
Kidney
Fig. 4. The effect of AMT on the glutathione peroxidase (GPx) activity in goldfish liver and kidney. Other information as in Fig. 1.
after 24 h of AMT treatment but this was reversed by 168 h. TBARS levels often change very little under various conditions that clearly stimulate oxidative stress development; for example, TBARS levels were not altered in some frog species under oxidative stress arising during recovery from anoxia, freezing or hibernation (Storey, 1996; Bagnyukova et al., 2003). The aforementioned work suggests that species that are adapted to survive in anoxic or hypoxic conditions cope efficiently with oxidative damages to membranes. Our data support this idea indicating that after an initial imbalance that resulted in TBARS accumulation, the organs readjusted in an adaptive phase that returned TBARS levels to control values by 168 h. Moreover, a short-term increase in lipid peroxidation products which are the result of oxidative stress was proposed to trigger enhanced protection against stressful conditions (Baraboy and Sutkovoy, 1997). These products as well as H2O2 alone can be involved in the regulation of the adaptive response via ROS-sensitive transcription factors such as AP-1 or NF-nB (Fandrey et al., 1994; Lee and Choi, 2003). One of targets of this regulation may be antioxidant enzymes. Disruption of the prooxidant/antioxidant balance due to depletion of one of antioxidant enzyme often results in compensatory changes in other enzyme activities as well as in contents of low-molecular weight antioxidants (Michiels et al., 1994). For instance, long-term inhibition of catalase led to an induction of enzymatic (SOD, GR) and nonenzymatic (glutathione, ascorbate) antioxidants in frog liver and kidney (Lopez-Torres et al., 1993). Treatment with AMT increased glutathione levels in rat brain (Benzi et al., 1990) and induced glutathione synthesis and SOD activity in Musca domestica (Allen et al., 1983). On the other hand, it is known that antioxidant enzymes themselves are very sensitive to oxidative inactivation by ROS (Szweda and Stadtman, 1992; HermesLima and Storey, 1993). Therefore, the net change in antioxidant enzyme activities under oxidative stress is the result of these two oppositely directed processes. Catalase inhibition by AMT did not affect SOD activity in liver or kidney of any of the experimental groups of goldfish
(data not shown) whereas activities of glutathione-dependent enzymes were influenced. GPx activity in liver was reduced by 60% after 24 h of AMT treatment but returned to the initial level after 168 h (Fig. 4). In kidney, GPx activity was increased after 168 h, by about 1.5-fold in saline-treated fish and 2-fold in AMT-injected fish. GST activity in liver was not affected by experimental treatments but activity rose in kidney of both AMT groups by ¨1.8-fold as well as by a lesser amount in saline-injected fish sampled after 168 h (Fig. 5). GR activity showed only minor alterations under experimental conditions; the only significant effect was an ¨35% decrease in GR activity after 24 h in liver of fish injected by AMT (data not shown). G6PDH activity in liver decreased by about 50% in both AMT-injected groups and in the NaCl-injected group sampled after 168 h. In kidney G6PDH activities were not affected by any experimental treatment (data not shown). The data show that several of the antioxidant enzymes tested showed a ‘‘two-phase’’ response to catalase inhibition in liver. In the initial acute phase, after 24 h of AMT exposure, activities of GPx, GR and G6PDH in liver were reduced whereas in the longer term adaptive phase (168 h post-injection) activities were restored (except for G6PDH). Changes in the activities of these enzymes correlated inversely with TBARS levels; correlation coefficients (R 2) were 0.81, 0.78 and 0.65 for GPx, GR and G6PDH, respectively. This supports the idea that these enzymes might be inactivated by high levels of ROS generated as an initial response to catalase inhibition due either to elevated H2O2 itself or increased production of subsequent damage products such as lipid peroxides. Earlier studies showed that GPx activity decreased in AMT-treated frogs whereas TBARS levels increased (Barja de Quiroga et al., 1989). Two other enzymes, GR and G6PDH, are also inactivated by ROS (Worthington and Rosemeyer, 1976; Szweda and Stadtman, 1992). Renewed activities of all three enzymes in the adaptive phase suggest that de novo synthesis of these enzymes occurred. It was found also that in goldfish exposed to hyperoxia liver GST activities inversely correlated with TBARS levels (Lushchak et al., 2005). Apart from proposed above explanation, one may suggest that lipid 2000
GST, mU/mg protein
GPx, mU/mg protein
800
339
1600
1200 1
1,4 1
800
400
0
1
2 3
4
Liver
5
1
2
3
4
5
Kidney
Fig. 5. The effect of AMT on the glutathione-S-transferase (GST) activity in goldfish liver and kidney. Other information as in Fig. 1.
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GST, mU/mg protein
control values. Kidney has lower antioxidant enzyme activities and glutathione levels as compared to liver and in the adaptive phase showed a compensatory increase in GPx and GST activities. However, this did not prevent an accumulation of protein carbonyls. Hence, a lack of catalase may be counterbalanced by reorganization of other components of the antioxidant system. Transcriptional regulation via involving end products of lipid peroxidation may be a way to enhance protective mechanisms. However, in some cases, these compensatory responses do not entirely replace catalase function so that enhanced accumulation of selected oxidative damage products can occur.
y = -42,76x + 1053,8
900
R2 = 0,8625
700
500
300
Acknowledgements 3
7
11
15
Catalase, U/mg protein Fig. 6. The relationship between catalase activity and glutathione-S-transferase (GST) activity in goldfish kidney. Each point represents one experimental group (n = 5 – 7).
peroxidation products are involved in up-regulation of antioxidant defenses. In kidney, there were no changes in enzyme activities in the acute phase of AMT exposure except for an increase in GST activity. However, in the longer term adaptive phase, activities of both glutathione-metabolizing enzymes, GPx and GST, were elevated by about 2-fold as compared with controls. Interestingly, elevated activities of both enzymes were also found in the saline-injected group sampled at 168 h although the increase was smaller than in AMT-treated fish. A correlation between GPx activity and CP levels was also found in kidney; these correlated positively with R 2 = 0.97. Kidney GST activity correlated inversely with catalase activity (R 2 = 0.86) (Fig. 6). Elevated GPx activity after 168 h of AMT treatment might be a compensatory response to catalase depletion. GST is involved in the detoxification of xenobiotics and aldehydic products of lipid peroxidation, such as 4-hydroxy-2-nonenal and malondialdehyde (Hermes-Lima, 2004). Therefore, both of these enzymes appear to be relevant to the adaptive response to reduced catalase activity, either by directly substituting for catalase depletion (GPx) or detoxifying damage to cellular lipids caused by enhanced ROS levels (GST). Generally, AMT injection caused a disturbance of cellular metabolism in goldfish organs which was confirmed by elevated levels of CP and TBARS as well as changes in protein concentration. The response of the antioxidant enzymes tested to suppressed catalase activity developed in a timedependent manner showing decreased or unchanged activities in the acute (24 h) phase and restored or elevated activities in the adaptive (168 h) phase. The patterns of the activity changes were organ specific. Goldfish liver possesses a powerful antioxidant potential (Lushchak et al., 2001; this study) and appears to successfully cope with catalase depletion; hence, after 7 days the levels of oxidative stress markers (CP, TBARS) as well as the antioxidant enzyme activities were restored to
We are grateful to J.M. Storey for critical reading of the manuscript. Special gratitude is to anonymous referees’ recommendations of which helped to improve the manuscript. Supported in part by a discovery grant from the Natural Sciences and Engineering Research Council of Canada to KBS and a grant from the Ministry of Education and Science of Ukraine to VIL. References Allen, R.G., Farmer, K.J., Sohal, R.S., 1983. Effect of catalase inactivation on levels of inorganic peroxides, superoxide dismutase, glutathione, oxygen consumption and life span in adult houseflies (Musca domestica). Biochem. J. 216, 503 – 506. Bagnyukova, T.V., Storey, K.B., Lushchak, V.I., 2003. Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation. J. Therm. Biol. 28, 21 – 28. Baraboy, V.A., Sutkovoy, D.A., 1997. Oxidative-Antioxidant Homeostasis at Norm and Pathology. Chernobylinterinform, Kyiv. Barja de Quiroga, G., Gil, P., Lopez-Torres, M., 1988. Physiological significance of catalase and glutathione peroxidases, and in vivo peroxidation, in selected tissues of the toad Discoglossus pictus (Amphibia) during acclimation to normobaric hyperoxia. J. Comp. Physiol. 158B, 583 – 590. Barja de Quiroga, G., Lopez-Torres, M., Perez-Campo, R., 1989. Catalase is needed to avoid tissue peroxidation in Rana perezi in normoxia. Comp. Biochem. Physiol. C 94, 391 – 398. Benzi, G., Marzatico, F., Pastoris, O., Villa, R.F., 1990. Influence of oxidative stress on the age-linked alterations of the cerebral glutathione system. J. Neurosci. Res. 26, 120 – 128. Bouzyk, E., Iwanenko, T., Jarocewicz, N., Kruszewski, M., Sochanowicz, B., Szumiel, I., 1997. Antioxidant defense system in differentially hydrogen peroxide sensitive L5178Y sublines. Free Radic. Biol. Med. 22, 697 – 704. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 2, 248 – 254. Brooks, S.P.J., 1992. A simple computer program with statistical tests for the analysis of enzyme kinetics. BioTechniques 13, 906 – 911. Dorval, J., Hontela, A., 2003. Role of glutathione redox cycle and catalase in defense against oxidative stress induced by endosulfan in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicol. Appl. Pharmacol. 192, 191 – 200. Fandrey, J., Frede, S., Jelkmann, W., 1994. Role of hydrogen peroxide in hypoxia-induced erythropoietin production. Biochem. J. 303, 507 – 510. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radicals in Biology and Medicine. Clarendon Press, Oxford. Hermes-Lima, M., 2004. Oxygen in biology and biochemistry: role of free radicals. In: Storey, K.B. (Ed.), Functional Metabolism: Regulation and Adaptation. Wiley-Liss, Hoboken, NJ, pp. 319 – 368.
T.V. Bagnyukova et al. / Comparative Biochemistry and Physiology, Part B 142 (2005) 335 – 341 Hermes-Lima, M., Storey, K.B., 1993. In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile. Mol. Cell. Biochem. 124, 149 – 158. Hiraishi, H., Terano, A., Ota, S., Mutoh, H., Sugimoto, M., Razandi, M., Ivey, K.J., 1991. Antioxidant defenses of cultured gastric cells against oxygen metabolites: role of GSH redox cycle and endogenous catalase. Am. J. Physiol. 261, G921 – G928. Kehrer, J.P., 2000. The Haber – Weiss reaction and mechanisms of toxicity. Toxicology 149, 43 – 50. Lee, P.J., Choi, A.M.K., 2003. Pathways of cell signaling in hyperoxia. Free Radic. Biol. Med. 35, 341 – 350. Lenz, A.-G., Costabel, U., Shaltiel, S., Levine, R.L., 1989. Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride. Anal. Biochem. 177, 419 – 425. Lopez-Torres, M., Perez-Campo, R., Rojas, C., Cadenas, S., Barja, G., 1993. Simultaneous induction of SOD, glutathione reductase, GSH, and ascorbate in liver and kidney correlates with survival during aging. Free Radic. Biol. Med. 15, 133 – 142. Lushchak, V.I., Lushchak, L.P., Mota, A.A., Hermes-Lima, M., 2001. Oxidative stress and antioxidant defenses in goldfish Carassius auratus during anoxia and reoxygenation. Am. J. Physiol. 280, R100 – R107. Lushchak, O.V., Bagnyukova, T.V., Lushchak, V.I., 2003. Effect of aminotriazole on the activity of catalase and glucose-6-phosphate dehydrogenase of two frog species, Rana ridibunda` and R. esculenta. Ukr. Biochem. J. 75, 45 – 50. Lushchak, V.I., Bagnyukova, T.V., Husak, V.V., Luzhna, L.I., Lushchak, O.V., Storey, K.B., 2005. Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues. Int. J. Biochem. Cell Biol. 37, 1670 – 1680.
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Margoliash, E., Novogrodsky, A., Schejter, A., 1960. Irreversible reaction of 3amino-1,2,4-triazole and related inhibitors with the protein of catalase. Biochem. J. 74, 339 – 350. Michiels, C., Raes, M., Toussaint, O., Remacle, J., 1994. Importance of Seglutathione peroxidase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress. Free Radic. Biol. Med. 17, 235 – 248. Remacle, J., Lambert, D., Raes, M., Pigeolet, E., Michiels, C., Toussaint, O., 1992. Importance of various antioxidant enzymes for cell stability. Confrontation between theoretical and experimental data. Biochem. J. 286, 41 – 46. Rice-Evans, C.A., Diplock, A.T., Symons, M.C.R., 1991. Techniques in free radical research. In: Burton, R.H., Knippenberg, P.H. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology. Elsevier, Amsterdam, pp. 147 – 149. Sies, H., 1991. Oxidative stress: introduction. In: Sies, H. (Ed.), Oxidative Stress: Oxidants and Antioxidants. Academic Press, San Diego, pp. 21 – 48. Stadtman, E.R., Levine, R.L., 2000. Protein oxidation. Ann. N. Y. Acad. Sci. 899, 191 – 208. Storey, K.B., 1996. Oxidative stress: animal adaptations in nature. Braz. J. Med. Biol. Res. 29, 1715 – 1733. Szweda, L.I., Stadtman, E.R., 1992. Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Structural and functional changes. J. Biol. Chem. 267, 3096 – 3100. van den Thillart, G., van Waarde, A., 1985. Teleosts in hypoxia: aspects of anaerobic metabolism. Mol. Physiol. 8, 393 – 409. Worthington, D.J., Rosemeyer, M.A., 1976. Glutathione reductase from human erythrocytes. Eur. J. Biochem. 67, 231 – 238.
Adaptive response of antioxidant enzymes to catalase inhibition by aminotriazole in goldfish liver and kidney Tetyana V. Bagnyukova a, Kenneth B. Storey b, Volodymyr I. Lushchak a,* a
Department of Biochemistry, Precarpathian National University named after Vassyl Stefanyk, 57 Shevchenko Str., 76025, Ivano-Frankivsk, Ukraine b Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada K1S 5B6 Received 7 April 2005; received in revised form 17 August 2005; accepted 18 August 2005 Available online 15 September 2005
Abstract This study was undertaken to clarify the physiological role of catalase in the maintenance of pro/antioxidant balance in goldfish tissues by inhibiting the enzyme in vivo with 3-amino 1,2,4-triazole. Intraperitoneal injection of aminotriazole (0.5 mg/g wet mass) caused a decrease in liver catalase activity by 83% after 24 h that was sustained after 168 h post-injection. In kidney catalase activity was reduced by ¨50% and 70% at the two time points, respectively. Levels of protein carbonyls were unchanged in liver but rose by 2-fold in kidney after 168 h. Levels of thiobarbituric acid-reactive substances were elevated in both tissues after 24 h but were reversed by 168 h. Glutathione peroxidase and glutathione-S-transferase activities increased in kidney after aminotriazole treatment whereas activities of glutathione peroxidase and glutathione reductase in liver decreased after 24 h but rebounded by 168 h. Liver glucose-6-phosphate dehydrogenase activity was reduced at both time points. Activities of these three enzymes in liver correlated inversely with the levels of lipid damage products (R 2 = 0.65 – 0.81) suggesting that they may have been oxidatively inactivated. Glutathione-S-transferase activity also correlated inversely with catalase (R 2 = 0.86). Hence, the response to catalase depletion involves compensatory changes in the activities of enzymes of glutathione metabolism. D 2005 Elsevier Inc. All rights reserved. Keywords: 3-Amino 1,2,4-triazole; Carassius auratus; Carbonyl proteins; Glutathione peroxidase; Glutathione-S-transferase; Kidney; Liver; Thiobarbituric reactive substances
1. Introduction All aerobic organisms need molecular oxygen for their oxidative metabolic processes but as a consequence must also deal with the formation of dangerous reactive oxygen species (ROS) including superoxide anion (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (UOH) (Halliwell and Gutteridge, 1989). ROS attack and chemically alter cellular macromolecules including proteins, lipids and DNA causing metabolic damage that is often severe. Therefore, all organisms possess well-developed systems of antioxidant defense that include both low-molecular weight antioxidants and antioxidant enzymes. The main antioxidant enzymes are superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). Under normal physiological conditions there is the balance between ROS generation and their elimination by different * Corresponding author. Fax: +1 38 03422 31574. E-mail address: [email protected] (V.I. Lushchak). 1096-4959/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2005.08.003
antioxidant scavengers. If prooxidant processes are enhanced and/or the power of the antioxidant system decreased, oxidative stress arises (Sies, 1991). Although all protective components are needed to cope with oxidative stress, each particular enzyme accomplishes specific functions that can only be partly replaced by the actions of other antioxidant defenses. There are two enzymes that catabolize hydrogen peroxide. Catalase converts H2O2 to water and molecular oxygen, thus preventing the formation of extremely dangerous hydroxyl radical from H2O2 via the Fenton reaction (Kehrer, 2000). GPx degrades H2O2 using reduced glutathione. The cooperation between catalase and GPx in dealing with cellular H2O2 has been the subject of a number of investigations. In some studies, catalase seemed to be more important than GPx in detoxifying H2O2 (Barja de Quiroga et al., 1988, 1989; Bouzyk et al., 1997). Other studies, however, reported that GPx was more powerful in antioxidant defense (Hiraishi et al., 1991; Remacle et al., 1992; Dorval and Hontela, 2003). To elucidate the
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significance of an enzyme, specific inhibitors are widely used. For example, catalase can be irreversibly inhibited by 3-amino 1,2,4-triazole (AMT) (Margoliash et al., 1960). The goldfish, Carassius auratus L., is well adapted to varied oxygen concentrations, from hyperoxia to hypoxia and even anoxia (van den Thillart and van Waarde, 1985). This fish possesses potent antioxidant defenses, as confirmed by high antioxidant enzyme activities as well as glutathione levels, particularly in two metabolically active tissues, liver and kidney (Lushchak et al., 2001). These tissues are sites of xenobiotic detoxification and, therefore, are considered to be powerful ROS generators (Halliwell and Gutteridge, 1989). The aim of this study was to investigate the effect of catalase inhibition by AMT on markers of oxidative stress and the activities of selected antioxidant enzymes in liver and kidney in order to elucidate possible mechanisms that could compensate for catalase function when this enzyme was inhibited. 2. Materials and methods 2.1. Chemicals Phenylmethylsulfonyl fluoride (PMSF), butylated hydroxytoluene (BHT), yeast glutathione reductase (GR), 1-chloro-2,4dinitrobenzene (CDNB), reduced glutathione (GSH), oxidized glutathione (GSSG), glucose-6-phosphate (G6P), ethylenediamine-tetraacetic acid (EDTA), sodium azide, and 3-amino 1,2,4-triazole (AMT) were purchased from Sigma Chemical Co. (USA). N,N,NV,NV-tetramethylethylenediamine (TEMED), NADP+ and NADPH were from Reanal (Hungary), Tris – HCl was from BioRad, and guanidine – HCl from Fluka. All other reagents were of analytical grade. 2.2. Animals and experimental conditions Goldfish (C. auratusL.) of both sexes weighing 20 – 70 g were purchased at a local fish market (Ivano-Frankivsk, Ukraine) and were kept in dechlorinated tap water and fed with standard goldfish food. Temperature was maintained at 18 T 1 -C with a natural light– dark cycle with light from about 8:00 to 17:00 h. Goldfish were acclimated to these conditions for at least one month before experimentation. For preliminary investigation of the effect of AMT on catalase inhibition, fish were injected intraperitoneally with AMT diluted in physiological saline at final concentrations of 0.1, 0.5 or 1.0 mg/gram wet body mass (gww). The volume of injected solution was 0.45% of body weight. Sham-injected fish received 0.9% NaCl solution instead AMT solution. Fish were killed by transspinal dissection at 5, 10, 24, 48, 72, 120 and 168 h after the treatment and catalase activity was measured immediately in liver and kidney. For subsequent studies, fish were injected with AMT solution at a final concentration of 0.5 mg/gww (the volume was 0.3% of body mass). A sham-injected group was treated with the same volume of 0.9% NaCl. Control fish were not treated. After 24 or 168 h the fish injected with AMT or NaCl solutions were sampled for organ dissection. For sampling, fish were killed and
the liver and kidneys were quickly removed. The samples were immediately used to measure the parameters of interest. 2.3. Indices of oxidative stress Tissue samples were homogenized (1 : 10 w/v) using a Potter –Elvjeham glass homogenizer in 50 mM potassium phosphate (KPi) buffer, pH 7.0, containing 0.5 mM EDTA and a few crystals of PMSF, a protease inhibitor. A 250 AL aliquot of this homogenate was then mixed with 0.5 mL of 10% (final concentration) trichloroacetic acid (TCA) and centrifuged for 5 min at 13,000 g in a microcentrifuge. Levels of protein carbonyls (CP) were measured in the resulting pellets, and thiobarbituric acid reactive substances (TBARS) contents were assayed in the supernatants using a spectrophotometer SF-46 (LOMO, Russia). Carbonyl derivatives of proteins were detected by reaction with 2,4-dinitrophenylhydrazine (DNPH). Resulting 2,4-dinitrophenylhydrazones were quantified spectrophotometrically (Lenz et al., 1989) with minor changes to the assay procedure (Bagnyukova et al., 2003). Briefly, the pellets from TCA treated extracts (above) were incubated with 1 mL of 10 mM DNPH in 2 M HCl (samples) or 1 mL of 2 M HCl only (controls). After incubation, samples were centrifuged, and resulting pellets were washed three times with ethanol – ethylacetate (1 : 1, v/v). Pellets were then dissolved in 6 M guanidine – HCl. The amount of CP was evaluated spectrophotometrically at 370 nm using a molar extinction coefficient of 22I103 M 1 cm 1 (Lenz et al., 1989). The values were expressed as nanomoles of CP per milligram protein in the guanidine chloride solution. The decomposition of lipid hydroperoxides produces lowmolecular weight products, including malondialdehyde, which can be measured by the TBARS assay (Rice-Evans et al., 1991). The supernatants from the TCA extract (above) were combined with the same volume of TBA reagent, containing a saturated solution of TBA in 0.1 M HCl and 10 mM BHT previously dissolved in ethanol; pH was adjusted to 2.5. BHT was added to avoid tissue peroxidation during heating of the samples. Control samples contained water instead of supernatant. The mixtures were immersed in a boiling water bath for 60 min. After quick cooling, a volume of butanol equal to the mixture total volume was added and mixed vigorously. Samples were centrifuged for 10 min at 5000 g and the butanol phase was removed and used to evaluate the level of TBARS. Absorption was measured at 535 nm and a molar extinction coefficient of 156 103 M 1 cm 1 was used to calculate TBARS concentration (Rice-Evans et al., 1991). The values are expressed as nanomoles of TBARS per gram wet weight (gww) of tissue. 2.4. Assay of antioxidant enzyme activities Tissue homogenates prepared as for the TBARS/CP assay were centrifuged at 4 -C for 15 min at 15,000 g. Supernatants were removed and used for enzyme activity assays using a spectrophotometer SF-46 (LOMO, Russia). The activity of superoxide dismutase (SOD) was assayed as a function of its inhibitory action on quercetin oxidation
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(Bagnyukova et al., 2003). The reaction mixture contained (final concentrations): 30 mM Tris – HCl buffer (pH 10.0), 0.5 mM EDTA, 0.8 mM TEMED, 0.05 mM quercetin, and 1.5 –50 AL of supernatant. The reaction was monitored at 406 nm for 6 – 8 different volumes of supernatant. One unit of SOD activity is defined as the amount of enzyme (per milligram protein) that inhibits the quercetin oxidation reaction by 50% of maximal inhibition. In our case, the maximal inhibition was about 90%. Catalase activity was measured at 240 nm in a medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 10 mM H2O2, and 2– 100 AL of supernatant (Bagnyukova et al., 2003). Blanks were run without hydrogen peroxide and/or supernatant. Selenium-dependent glutathione peroxidase (GPx) activity was measured by a coupled assay linked with GR catalyzed oxidation of NADPH (Lushchak et al., 2001). A basal consumption of NADPH was recorded at 340 nm in a medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 0.25 mM NADPH, 4 mM sodium azide, 1 U/mL GR, 15 mM GSH, and 2 –50 AL of the supernatant. Then, 10 AL of H2O2 was added to a final concentration of 0.2 mM (final volume, 1 mL). Blanks contained no supernatant. Glutathione-S-transferase (GST) activity was measured by monitoring the formation of an adduct between GSH and CDNB at 340 nm (Lushchak et al., 2001). Reaction mixture contained 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 5 mM GSH, 1 mM CDNB and 2 – 7 AL of supernatant in a final volume of 1 mL. The reaction was initiated by the sequential addition of CDNB and supernatant. Blanks contained no CDNB. The activity of GR was assayed at 340 nm in medium containing 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 0.25 mM NADPH, 1 mM GSSG, and 40– 50 AL of supernatant (Bagnyukova et al., 2003). Blanks omitted GSSG. Glucose-6-phosphate dehydrogenase (G6PDH) activity was measured at 340 nm in 50 mM KPi-buffer (pH 7.0), 0.5 mM EDTA, 5 mM MgCl2, 0.2 mM NADP, and 2 mM G6P with 30 – 50 AL of supernatant (Bagnyukova et al., 2003). Blanks were run without G6P. One unit of catalase, GPx, GST, GR, or G6PDH activity is defined as the amount of the enzyme consuming 1 Amol of substrate or generating 1 Amol of product/ min; activities were expressed as international units (or milliunits) per milligram protein. 2.5. Protein measurements and statistics Protein concentration was measured by the Bradford method with Coomassie Brilliant Blue G-250 (Bradford, 1976) and using bovine serum albumin as a standard. Data are presented as means T SEM. Statistical analysis was performed using Student’s t-test. Inhibition values for SOD activity were calculated using an enzyme kinetics computer program (Brooks, 1992). Correlation analysis was performed using Excel software. 3. Results and discussion Goldfish liver and kidney have well-developed antioxidant systems that include both antioxidant enzymes and lowmolecular weight antioxidants such as glutathione. Goldfish
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liver shows particularly well-developed defenses with activities of catalase, GPx and GST that were 3- to 5-fold higher than in kidney whereas SOD, GR and G6PDH activities were similar in both tissues (Table 1). These values were similar to (catalase, GST) or lower than (GR, G6PDH) enzyme activities obtained in an earlier study that used vual tail goldfish (Lushchak et al., 2001). That study also showed that total glutathione content was 3.3-fold higher in goldfish liver than in kidney (Lushchak et al., 2001). It is known that AMT irreversibly inactivates catalase (Margoliash et al., 1960). A single AMT injection resulted in a quick decrease in catalase activity in frog liver and kidney and activity remained reduced over at least 6 days (Barja de Quiroga et al., 1989; Lushchak et al., 2003). In a preliminary study we administered AMT to goldfish at 0.1, 0.5 or 1.0 mg/ gww and measured its effects on catalase activity in liver and kidney at times ranging from 5 to 168 h after injection. AMT injection affected catalase activity in both dose- and timedependent manners (data not shown). Catalase activity decreased by more than 60% in liver within 5 h after injection with AMT (as compared with sham-injected animals) and remained at this level over the remainder of the experimental course. A similar result was found in kidney with the major decrease in enzyme activity by 50– 90% seen within the first 24 h of treatment; subsequently, activities remained low over the rest of the time course. Using these preliminary data, we chose treatment with AMT at a concentration of 0.5 mg/gww for 24 or 168 h as the design for a second set of experiments. Fig. 1 shows that injection with AMT at a dose of 0.5 mg/ gww caused a sharp decrease in catalase activity in liver. Activity had dropped to ¨17% of the value in control (uninjected) fish after 24 h and remained at this level after 168 h post-injection. In kidney, catalase activity was reduced by 50% after 24 h post-injection and by 66% after 168 h of treatment. Thus, a single AMT injection was enough to cause a strong decrease in organ catalase activities that lasted for at least 7 days. Sham injection of fish with physiological saline alone did not affect catalase activity in either liver or kidney; activities were not significantly different from control values at either 24 or 168 h post-injection. Experimental treatments caused some alterations in organ protein concentrations. Liver of fish sampled 168 h after injection showed a 50% increase in soluble protein content for both sham-injected and AMT-injected fish (Table 2). By contrast, protein content was slightly reduced in kidney after AMT treatment; a significant decrease of 16% was seen after 24 h and the level remained low at 168 h. Although we do not Table 1 Activities of antioxidant enzymes in liver and kidney of goldfish (n = 5 – 9) Enzyme
Liver
Kidney
SOD, U/mg Catalase, U/mg GPx, mU/mg GST, U/mg GR, mU/mg G6PDH, mU/mg
74.7 T 17.9 58.5 T 12.7 533 T 61 1.45 T 0.17 8.17 T 0.69 44.0 T 7.6
50.1 T16.9 13.1 T1.3 109 T 7 0.46 T 0.06 11.4 T 1.4 39.0 T 4.4
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16
1,4
CP, nmol/mg protein
60
40
20
0
1,2
1
2
3
1,4
4
5
Liver
1,2
1 2
3
12 1
8
4
1,4 4
0
5
1
2
Kidney
3
4
5
Liver
Fig. 1. The effect of AMT on catalase activity in goldfish liver and kidney. Experimental conditions are: (1) control uninjected fish, (2) 24 h post-injection with 0.9% NaCl, (3) 24 h post-injection with 0.5 mg/gww AMT, (4) 168 h postinjection with 0.9% NaCl (5) 168 h post-injection with 0.5 mg/gww AMT. Data are means T SEM, n = 5 – 7 fish. Numbers over the bars indicate significant differences from the indicated groups, P < 0.05.
know which types of proteins were affected, these changes may be considered as a general response to stress caused by saline or AMT. Catalase inhibition resulted in the development of oxidative stress in goldfish tissues which was confirmed by elevated levels of CP and TBARS in some cases. The amount of oxidatively damaged proteins, measured by CP levels, did not change in liver under any experimental condition but in kidney of AMT-treated fish CP content rose 2.4-fold after 168 h (Fig. 2). Levels of CP in kidney were also slightly elevated in shaminjected fish sampled at 168 h. Both tissues showed a similar pattern of change in TBARS levels, reflecting oxidative damage to lipids. TBARS content rose by 2- and 3-fold in liver and kidney, respectively, after 24 h post-injection but levels returned to control values by 168 h (Fig. 3). The different patterns of change in CP and TBARS levels probably reflect different cellular responses to oxidative damage to proteins versus lipids. Several explanations for this may be proposed. Firstly, cell proteins and lipids may have different susceptibilities to oxidation by ROS. Secondly, different defenses are available in cells to deal with oxidative damage to lipids and proteins. Lipid peroxides are decomposed by phospholipid Table 2 Soluble protein concentration (mg/mL) in liver and kidney of goldfish Groups
Liver
Kidney
Control 24 h NaCl 24 h AMT 168 h NaCl 168 h AMT
5.20 T 0.51 3.74 T 0.72 6.35 T 0.38* 7.97 T 0.74C 7.81 T 0.79C
4.19 T 0.27 4.07 T 0.40 3.53 T 0.22C 4.27 T 0.35 3.59 T 0.27
NaCl and AMT groups were injected with physiological saline or AMT, respectively, and sampled after 24 or 168 h. Protein was measured in the supernatants prepared for enzyme assay. C Significantly different from the corresponding control value or *from the group treated for 24 h with NaCl ( P < 0.05), n = 6 – 9.
1
2
3
4
5
Kidney
Fig. 2. The effect of AMT on protein carbonyl levels in goldfish liver and kidney. Other information as in Fig. 1.
hydroperoxide glutathione peroxidase (Hermes-Lima, 2004) whereas oxidized proteins are degraded by specific and nonspecific proteases whose activities depend, in turn, on a variety of factors and may be inhibited by ROS and their products (Stadtman and Levine, 2000). Thirdly, long-term differences in the accumulation and tolerance of different types of damaged macromolecules occur; for example, during aging and under prolonged stress conditions oxidized proteins tend to be accumulated (Stadtman and Levine, 2000). It appears that the inherent antioxidant defenses of goldfish liver were still able to protect hepatic proteins from oxidation damage even when catalase was depleted due to inhibition by AMT, a situation where intracellular H2O2 levels would presumably be very high. The greatly reduced activity of catalase might be compensated for in other ways, for example, by glutathione-linked enzymes. The same is true to a lesser extent for kidney where significant CP accumulation was seen only after 168 h. With respect to TBARS content, liver and kidney showed an early sharp increase, by two- to three-fold, 80 1,2
TBARS, nmol/gww
Catalase, U/mg protein
80
60
1,2
1
40
20
0
1
2
3
Liver
4
5
1 2
3
4
5
Kidney
Fig. 3. The effect of AMT on the levels of thiobarbituric acid-reactive substances in goldfish liver and kidney. Other information as in Fig. 1.
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600
400 1,4
1,2 1
200
0 1
2
3
4
Liver
5
1
2
3
4
5
Kidney
Fig. 4. The effect of AMT on the glutathione peroxidase (GPx) activity in goldfish liver and kidney. Other information as in Fig. 1.
after 24 h of AMT treatment but this was reversed by 168 h. TBARS levels often change very little under various conditions that clearly stimulate oxidative stress development; for example, TBARS levels were not altered in some frog species under oxidative stress arising during recovery from anoxia, freezing or hibernation (Storey, 1996; Bagnyukova et al., 2003). The aforementioned work suggests that species that are adapted to survive in anoxic or hypoxic conditions cope efficiently with oxidative damages to membranes. Our data support this idea indicating that after an initial imbalance that resulted in TBARS accumulation, the organs readjusted in an adaptive phase that returned TBARS levels to control values by 168 h. Moreover, a short-term increase in lipid peroxidation products which are the result of oxidative stress was proposed to trigger enhanced protection against stressful conditions (Baraboy and Sutkovoy, 1997). These products as well as H2O2 alone can be involved in the regulation of the adaptive response via ROS-sensitive transcription factors such as AP-1 or NF-nB (Fandrey et al., 1994; Lee and Choi, 2003). One of targets of this regulation may be antioxidant enzymes. Disruption of the prooxidant/antioxidant balance due to depletion of one of antioxidant enzyme often results in compensatory changes in other enzyme activities as well as in contents of low-molecular weight antioxidants (Michiels et al., 1994). For instance, long-term inhibition of catalase led to an induction of enzymatic (SOD, GR) and nonenzymatic (glutathione, ascorbate) antioxidants in frog liver and kidney (Lopez-Torres et al., 1993). Treatment with AMT increased glutathione levels in rat brain (Benzi et al., 1990) and induced glutathione synthesis and SOD activity in Musca domestica (Allen et al., 1983). On the other hand, it is known that antioxidant enzymes themselves are very sensitive to oxidative inactivation by ROS (Szweda and Stadtman, 1992; HermesLima and Storey, 1993). Therefore, the net change in antioxidant enzyme activities under oxidative stress is the result of these two oppositely directed processes. Catalase inhibition by AMT did not affect SOD activity in liver or kidney of any of the experimental groups of goldfish
(data not shown) whereas activities of glutathione-dependent enzymes were influenced. GPx activity in liver was reduced by 60% after 24 h of AMT treatment but returned to the initial level after 168 h (Fig. 4). In kidney, GPx activity was increased after 168 h, by about 1.5-fold in saline-treated fish and 2-fold in AMT-injected fish. GST activity in liver was not affected by experimental treatments but activity rose in kidney of both AMT groups by ¨1.8-fold as well as by a lesser amount in saline-injected fish sampled after 168 h (Fig. 5). GR activity showed only minor alterations under experimental conditions; the only significant effect was an ¨35% decrease in GR activity after 24 h in liver of fish injected by AMT (data not shown). G6PDH activity in liver decreased by about 50% in both AMT-injected groups and in the NaCl-injected group sampled after 168 h. In kidney G6PDH activities were not affected by any experimental treatment (data not shown). The data show that several of the antioxidant enzymes tested showed a ‘‘two-phase’’ response to catalase inhibition in liver. In the initial acute phase, after 24 h of AMT exposure, activities of GPx, GR and G6PDH in liver were reduced whereas in the longer term adaptive phase (168 h post-injection) activities were restored (except for G6PDH). Changes in the activities of these enzymes correlated inversely with TBARS levels; correlation coefficients (R 2) were 0.81, 0.78 and 0.65 for GPx, GR and G6PDH, respectively. This supports the idea that these enzymes might be inactivated by high levels of ROS generated as an initial response to catalase inhibition due either to elevated H2O2 itself or increased production of subsequent damage products such as lipid peroxides. Earlier studies showed that GPx activity decreased in AMT-treated frogs whereas TBARS levels increased (Barja de Quiroga et al., 1989). Two other enzymes, GR and G6PDH, are also inactivated by ROS (Worthington and Rosemeyer, 1976; Szweda and Stadtman, 1992). Renewed activities of all three enzymes in the adaptive phase suggest that de novo synthesis of these enzymes occurred. It was found also that in goldfish exposed to hyperoxia liver GST activities inversely correlated with TBARS levels (Lushchak et al., 2005). Apart from proposed above explanation, one may suggest that lipid 2000
GST, mU/mg protein
GPx, mU/mg protein
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339
1600
1200 1
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800
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0
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2 3
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Fig. 5. The effect of AMT on the glutathione-S-transferase (GST) activity in goldfish liver and kidney. Other information as in Fig. 1.
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GST, mU/mg protein
control values. Kidney has lower antioxidant enzyme activities and glutathione levels as compared to liver and in the adaptive phase showed a compensatory increase in GPx and GST activities. However, this did not prevent an accumulation of protein carbonyls. Hence, a lack of catalase may be counterbalanced by reorganization of other components of the antioxidant system. Transcriptional regulation via involving end products of lipid peroxidation may be a way to enhance protective mechanisms. However, in some cases, these compensatory responses do not entirely replace catalase function so that enhanced accumulation of selected oxidative damage products can occur.
y = -42,76x + 1053,8
900
R2 = 0,8625
700
500
300
Acknowledgements 3
7
11
15
Catalase, U/mg protein Fig. 6. The relationship between catalase activity and glutathione-S-transferase (GST) activity in goldfish kidney. Each point represents one experimental group (n = 5 – 7).
peroxidation products are involved in up-regulation of antioxidant defenses. In kidney, there were no changes in enzyme activities in the acute phase of AMT exposure except for an increase in GST activity. However, in the longer term adaptive phase, activities of both glutathione-metabolizing enzymes, GPx and GST, were elevated by about 2-fold as compared with controls. Interestingly, elevated activities of both enzymes were also found in the saline-injected group sampled at 168 h although the increase was smaller than in AMT-treated fish. A correlation between GPx activity and CP levels was also found in kidney; these correlated positively with R 2 = 0.97. Kidney GST activity correlated inversely with catalase activity (R 2 = 0.86) (Fig. 6). Elevated GPx activity after 168 h of AMT treatment might be a compensatory response to catalase depletion. GST is involved in the detoxification of xenobiotics and aldehydic products of lipid peroxidation, such as 4-hydroxy-2-nonenal and malondialdehyde (Hermes-Lima, 2004). Therefore, both of these enzymes appear to be relevant to the adaptive response to reduced catalase activity, either by directly substituting for catalase depletion (GPx) or detoxifying damage to cellular lipids caused by enhanced ROS levels (GST). Generally, AMT injection caused a disturbance of cellular metabolism in goldfish organs which was confirmed by elevated levels of CP and TBARS as well as changes in protein concentration. The response of the antioxidant enzymes tested to suppressed catalase activity developed in a timedependent manner showing decreased or unchanged activities in the acute (24 h) phase and restored or elevated activities in the adaptive (168 h) phase. The patterns of the activity changes were organ specific. Goldfish liver possesses a powerful antioxidant potential (Lushchak et al., 2001; this study) and appears to successfully cope with catalase depletion; hence, after 7 days the levels of oxidative stress markers (CP, TBARS) as well as the antioxidant enzyme activities were restored to
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