Journal of Neurochemistry Raven Press, Ltd., New York 0 1992 International Society for Neurochernistry
Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities in Copper/Zinc-Superoxide Dismutase Transgenic Mice *tS. Przedborski, *V. Jackson-Lewis, *V. Kostic, SE. Carlson, SC. J. Epstein, and *J. L. Cadet 'Laboralory of Preclinical Neurosciences, Department of Neurology, College of Physicians & Surgeons, Columbia University, New York, New York; +Departments of Pediatrics and of Biochemistry and Biophysics, University of California, San Francisco, California, U.S.A.;and ?Department of Neurology, H6pital Erasme, Universite'Libre de Bruxelles, Bruxelles, Belgium
greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSHPx activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn-SOD activity, SOD-transgenic mice also exhibit increases in other enzymes that scavenge oxygen-based radicals. We also found that aminotriazole caused significantlygreater inhibition of brain catalase activity in SOD-transgenic mice (67.6%) than in nontransgenic littermates (43.6%). The latter finding provides evidence for a higher production of H202in the brains of SOD-transgenic mice. When taken together, these results further support the use of these animals to assess the role of free radicals in ischemia/reperfusion and in the aging process. Key Words: Transgenic mice-Superoxide dismutase-Catalase-Glutathione peroxidase-Free radicals-Brain. Przedborski S. et al. Superoxide dismutase, catalase, and glutathione peroxidase activities in copper/zinc-superoxide dismutase transgenic mice. J. Neurochem. 58, 1760-1767 (1992).
Abstract: Copperlzinc-superoxide dismutase (CuZn-SOD) transgenic mice overexpress the gene for human CuZn-SOD. To assess the effects of the overexpression of CuZn-SOD on the brain scavenging systems, we have measured the activities of manganese-SOD (Mn-SOD), catalase, and glutathione peroxidase (GSH-Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn-SOD activity was highest in the caudate-putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn-SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42-fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn-SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)-transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed 4 0 % of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the
been implicated in several neurotoxic and neuropathologic events (Halliwell and Gutteridge, 1985~;Cadet, 1988). The effects of free radicals in the brain are thought to be triggered by its high consumption of oxygen and its high concentration of phospholipids,which can propagate free radical-generated reactions. These facts led to the suggestions that active oxygen species
may play a role in the developmental pathobiology of Parkinson's disease (Cohen, 1984; Dexter et al., 1989; Saggu et al., 1989), ischemic or traumatic brain injury (Chan, 1989; Chan et al., 1991), tardive dyskinesias (Cadet et al., 1986), and aging (Halliwell and Gutteridge, 19856). Several enzymes act in concert to protect the host from the ravages caused by these active oxygen species (Freeman and Crapo, 1982). These include catalase (EC 1.1 1.1.6) and glutathione peroxidase (GSH-
Received July 25, 1991; revised manuscript received October 8, I99 1 ;accepted October 16, 199 1. Address correspondence and reprint requests to Dr. J. L. Cadet at
Abbreviations used: CuZn-SOD, copper/zinc-superoxidedismutase; GSH-Px, glutathione peroxidase; Mn-SOD, manganese-superoxide dismutase; 0 2 - , superoxide radical; OH, hydroxyl radical; SOD,
Department of Neurology, Columbia University College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032, U.S.A.
H202, and hydroxyl radicals
( OH) are oxygen-based reactive compounds that have
SCA VENGER ENZYMES IN Cu/Zn-SOD TRANSGENIC MICE Px; EC 1.1 1.1 .9), which break down H202, and the 02--scavenging enzyme superoxide dismutase (SOD; EC 22.214.171.124) (Freeman and Crapo, 1982). SOD is a metalloenzyme that is essential to the dismutation of 02-to H202(Fridovitch, 1986). SOD is a very important component of the cellular defense against oxygen toxicity because 0 2 - can participate with H202 in the iron-catalyzed Haber-Weiss reaction to generate devastatingly reactive OH radicals (Freeman and Crapo, 1982). It has even been suggested that most of the toxicity of H202is due to its reduction to the * OH radical by metal-catalyzed reactions (Badwey and Karnovsky, 1980). To test the role of SOD gene dosage in the pathobiology of Down’s syndrome, Epstein et al. (1987) have constructed SOD-transgenic mice that overexpress the human copper/zinc-SOD (CuZn-SOD) gene. It was thought, in addition, that these mice might serve as a good animal model to test the contribution of free radicals in the deleterious effects of some neurotoxic agents (Cadet et al., 1990; Chan et al., 1990; Przedborski et al., 1990). To clarify the role of SOD in this model, it is important to know if other scavenging enzymes are also affected in these SOD-transgenic mice. Thus, we have quantified the activity of CuZn-SOD, manganeseSOD (Mn-SOD), catalase, and GSH-Px in both cytosolic and particulate fractions of various brain regions of SOD-transgenic mice and their nontransgenic littermates. In addition, because the brain contains relatively low specific activities of H202-scavenging enzymes (Sinet et al., 1980), we also compared the concentration of H202(Sinet et al., 1980) in the brains of SOD-transgenic mice and their nontransgenic littermates to test the possibility that H202production might be increased in the SOD-transgenic animals secondary to increased 02-dismutation. MATERIALS AND M E T H O D S Animals Transgenic mice of strain HS/SF-2 18 carrying the human CuZn-SOD gene, produced as described by Epstein et al. (1 987), were used. The genome of this strain carries several copies on the human CuZn-SOD gene, presumably in tandem array. The founder mice have been bred to produce transgenic offspring expressing the human CuZn-SOD gene. Transgenic mice were identified by northern blot analysis for the presence of the mRNA of the human CuZn-SOD gene (Epstein et al., 1987; Chan et al., 1991). Preparation of samples White female SOD-transgenic mice (n = 12) and their nontransgenic littermates (n = 12), both groups weighing 25-30 g, were killed by decapitation. The brains were rapidly removed from the skull and thoroughly rinsed in ice-cold saline. Thereafter, frontal cortex, caudate-putamen complex, hippocampus, cerebellum, and brainstem were dissected out on ice and frozen immediately on dry ice. To quantify brain SOD isoenzymes and catalase and GSH-Px activities in both cytosolic and particulate fractions, brain samples were weighed and homogenized by hand with a Teflon-glass ho-
mogenizer in 30 volumes of ice-cold 0.1 Mphosphate buffer (pH 7.8) containing 0.1 mM EDTA. The homogenates were then centrifuged at 100,000 g for 60 rnin at 4°C. These first supernatants, which correspond to cytosolic fractions, were collected, immediately frozen, and kept at -80°C until assayed. The pellets were resuspended in fresh ice-cold buffer (half of the initial volume), frozen, thawed three times, and then centrifuged at 100,000gfor 60 rnin at 4°C. These second supernatants, which correspond to the particulate fraction, were removed, frozen, and also stored at -80°C until assayed. On the day of the assay, 5-ml plastic syringes were packed with presoaked Sephadex G- 10 and were centrifuged at 160 g for 2 min to remove excess buffer. An aliquot (0.5 ml) of the samples was loaded on the Sephadex column and centrifuged at 160 g for 2 min. Thereafter, 0.5 ml of buffer was added to the column and centrifuged as before. In preliminary experiments, we showed that this procedure using Sephadex G-10 columns did not result in any loss of enzymatic activity. The quality of purification was similar to that obtained using dialysis techniques (Perumal et al., 1989). The elution volume (purified sample) was collected, and scavenging enzymes were assayed colorimetrically using a Shimadzu UV- 160 spectrophotometer. Assays of scavenger enzyme activities SOD activity was determined by the NADH-nitroblue tetrazolium assay at 540 nm according to the method described by Fried (1975) with minor modifications (Perumal et al., 1989; Przedborski et al., 1991). All experiments designed to show the activity of the CuZn and the Mn forms of SOD were carried out with and without inclusion of 5 mM KCN in the incubation mixture (Ledig et al., 1982); the samples were incubated at room temperature for 30 min in the incubation mixture before the reaction was started to ensure complete inhibition of the CuZn-SOD (Bize et al., 1980). Catalase activity was determined according to the method described by Aebi (1984) based on H202decomposition followed at 240 nm. GSH-Px activity was determined according to the method described by Hohe and Gunzler (1984) based on NADPH oxidation followed at 340 nm. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The method of Cross et al. (1979) was used to assess the contribution of blood contamination in brain SOD, catalase, and GSH-Px activities. Aminotriazole treatment Transgenic (n = 3 ) and nontransgenic (n = 3 ) mice were injected intraperitoneally with 1 g/kg of 3-amino-1,2,4-triazole (aminotriazole; Sigma, St. Louis, MO, U.S.A.) in saline; transgenic (n = 3) and nontransgenic (n = 3) control mice received the same volume of saline solution. One hour after the injection, the mice were killed by decapitation, and their brains were processed as described above; this time point was selected based on the study of Sinet et al. (1980), who showed that under these conditions there is an -50% inhibition in catalase activity. Catalase activity was measured in different brain regions of the four groups of animals as described above. To quantify aminotriazole content, 0.5 ml of 1 M trichloroacetic acid was added to 1.0 ml of uncentrifuged brain homogenates. Aminotriazole content of homogenates was measured colorimetrically(Green and Feinstein, 1957; Yusa et al., 1987) by diazotization and coupling with disodium 4,5-dihydroxy-2,7-naphthalenedisulfonic acid (Sigma).
J. Neurochem., Vol. 58, No. 5, 1992
S. PRZEDBORSKI ET AL.
a small KCN-sensitive SOD activity that remained unchanged after three washes of the pellet used for the preparation of the particulate fraction; this indicates that this activity. is not due to contamination of the particulate fraction by enzyme from the cytosol and that a small amount of CuZn-SOD activity does exist in the particulate fractions, most likely originatingfrom cell nuclei and lysosomes (Slot et al., 1986), peroxisomes (Keller et al., 1991), and mitochondria (Weisiger and Fridovich, 1973). This KCN-sensitive activity found in the particulate fractions was 3.9 1-4.84-fold higher in the brain regions of SOD-transgenic mice in comparison with their nontransgenic littermates (Table 2). Mn-SOD activity did not show any significant differences in its regional distribution. Unexpectedly, the particulate Mn-SOD activity was significantly higher in SOD-transgenic mice compared with their nontransgenic littermates (Table 2). These differences remained unchanged after the assays were repeated with higher concentrations of KCN (10 instead of 5 mM).
statistics All the assays were camed out in duplicates. For the regional distribution of scavenging enzymes activities, differences between transgenic and nontransgenic mice and between brain regions were tested by a two-way analyses of variance, whereas for the aminotriazole experiment, a threeway analysis of variance was used. In all cases, the analysis of variance was followed by a ScheE post hoc test. For the blood scavenging enzyme activities, differences between transgenic and nontransgenic mice were tested by a two-tailed Student's t test. In all cases the null hypothesis was rejected at the 0.05 level. All data are expressed as mean & SEM values.
RESULTS SOD isoenzyme activities in mouse brain In the cytosolic fraction, addition of 5 mM KCN to the incubation mixture caused a marked reduction in SOD activity (Table 1). The KCN-sensitive SOD activity, which represents the CuZn-SOD enzyme, showed significant differences among the various regions of the brain in both groups of mice (Table 1). For instance, the highest CuZn-SOD activities were found in the caudate-putamen complex, followed by the brainstem and the hippocampus; the lowest activities were measured in the cerebellum (Table 1). C a n SOD activities were two to 3.42 times higher in the subregions of the brains of SOD-transgenic mice in comparison with their nontransgenic littermates(Table 1). Cytosolic KCN-resistant SOD activities, which represent Mn-SOD, were low in comparison with CuZnSOD activities (see Table 1). These values were not modified by either a second centrifugation of the cytosolic fraction or an increase in KCN concentration (10 a) There . were no significant differences in MnSOD activities either between brain regions or between SOD-transgenic and nontransgenic mice (Table 1). In the particulate fractions, most of the SOD activities were KCN resistant; this indicates that mostly MnSOD is found in that fraction. This is in contrast to the situation in the cytosol. We were able to document
Catalase and GSH-Px activities in brain regions More than 80%of the catalase activity was found in the cytosolic fractions (Table 3). Although the exact subcellular distribution of catalase is still not entirely defined (Noh1and Hegner, 1978),it is known that peroxisomes contain copious amounts of this enzyme (Gaunt and DeDuve, 1976; Keller et al., 199I). Thus, it is likely that the high cytosolic activity found in our study is an artifact of tissue preparation. Except for the frontal cortex, catalase activity was evenly distributed in the different brain regions tested in both groups of mice (Table 3). Cytosolic catalase activity was higher in all subregions of the brain of SOD-transgenic mice compared with their nontransgenic littermates (Table 3). Furthermore, there was a strong positive linear correlation between cytosolic KCN-sensitive SOD activity and catalase activity among both groups of mice ( r = 0.71, p < 0.0001). As for catalase, almost all of the brain GSH-Px activity was found in cytosolic fractions without there being any significant regional variations
TABLE 1. Cytosolic SOD activity in brain regions of SOD-transgenic mice and their nontransgenic littermates ~~
KCN-sensitive SOD activity
KCN-resistant SOD activity
FCx CPU Hip Cer BS
3.37 k 0.57 5.75 k 0.44 4.18 0.64 2.10 & 0.18 4.77 k 0.19
9.56 f 0.37' 13.39 & 0.52d 11.19 k 0.86' 7.20 & 0.8 1 9.56 It 0.44'
2.83 3.12 2.81 3.42 2.00
0.76 k 0.08 0.68 0.06 0.86 f 0.06 0.82 f 0.07 0.75 f 0.04
0.75 f 0.04 0.17 f 0.04 0.89 & 0.05 0.86 & 0.06 0.78 & 0.06
Ratio 0.98 1.13 1.03 1.04 I .05
SOD activity (U/mg of protein) was measured by a colorimetric procedure as described in Materials and Methods. One unit of SOD activity was defined as 50% inhibition of formazan formation. Data are mean f SEM values measured in duplicates in 12 SOD-transgenic and 12 nontransgenic mice. FCx,frontal cortex; CPu, caudate-putamen complex; Hip, hippocampus; Cer, cerebellum; BS, brainstem. SOD-transgenic/nontransgenic activity. ' p < 0.01, d p < 0.001 compared with nontransgenic mice by two-way analysis of variance followed by Scheffgs test.
J. Neurochem.. Vol. 58, No. 5. 1992
SCA VENGER ENZYMES IN Cu/Zn-SOD TRANSGENIC MICE
TABLE 2. Particulate SOD activity in brain regions of SODtransgmic mice and their nontransgmic littermates ~
KCN-sensitive SOD activity Brain area" FCx C h Hip CU
KCN-resistant SOD activity
0.055 f 0.033 0.082 f 0.018 0.079 f 0.025 0.065 ? 0.008 0.083 f 0.035
0.228 f 0.010' 0.330 f 0.015" 0.309 f 0.033" 0.272 f 0.010' 0.402 & 0.023"
4.14 4.03 3.9 I 4.19 4.84
0.88 f 0.07 0.87 ? 0.10 0.76 f 0.07 0.70 ? 0.10 0.78 f 0.09
1.50 f 0.07" 1.69 f 0.06' 1.73 f 0.10" 1.41 f O.Ogd 1.36 f 0.12'
1.70 1.94 2.28 2.00 1.74
SOD activity (U/mg of protein) was measured by a colorimetric procedure as described in Materials and Methods. One unit of SOD activity was defined as 50% inhibition of formazan formation. Data are mean f SEM values measured in duplicates in 12 SOD-transgenicand 12
nontransgenic mice. FCx, frontal cortex; CPu, caudateputamencomplex; Hip, hippocampus;Cer, cerebellum; BS,brainstem. SOD-transgenic/nontransgenicactivity. < 0.001, d p < 0.01, c p < 0.05 compared with nontransgenic mice by two-wayanalysis of variance followed by ScheWs test.
contamination. This range of blood contamination would have a negligible effect on the determination of activities of brain scavenging enzymes (see Tables 1-4).
(Table 4). In contrast to catalase activity, GSH-Px a o tivity was not different between the two groups of mice (Table 4).
Blood contamination of brain SOD, catalase,and GSH-Px activity The volume of blood present in brain homogenates was measured in the different regions studied and ranged from 0.024 &mg of protein of brain tissue in the caudate-putamen complex to 0.040 &mg of protein of brain tissue in the brainstem. No significant differences in blood contamination of the brain homogenates were found between the SOD-transgenic and nontransgenic mice. Blood total SOD, catalase, and GSH-Px activities were measured in SOD-transgenic and nontransgenic mice (Table 5). In the blood, total SOD activity was 1.9 times higher in SOD-transgenic mice than in their nontransgenic littermates, whereas catalase and GSH-Px activities were not significantly different between the two groups of animals (Table 5). These enzymatic activitiesare in good agreement with published values (Maral et d.,1977; EpStein et al., 1987). Therefore, (0.02 U/mg of protein of total SOD, t0.04 U/mg of protein of catalase, and (0.002 U/mg of protein of GSH-Px were attributed to blood
Effects of amhtriazole on catdase activity in brain regions One hour after the intraperitoneal injection (1 g/kg body weight), the aminotriazole content of the brain did not show any significant regional differences; it ranged from 2.82 to 3.24 pg/mg of protein. No significant differencesin aminotriazole content were found between the SOD-transgenic and nontransgenic . . mice. In nontransgenicmice, aminotriazoleadmmstmtion caused a marked inhibition in brain catalase activity compared with saline-injected controls, without any noticeable differences between the various regions of the brain examined; the mean inhibition was 43.6% (Fig. 1). In SOD-tmnsgenicmice, aminotriazole c a u s e d greater inhibition in brain catalase activity compared with saline-injected controls. There were also no significant differences in catalase inhibition between the various regions of the brain studid, the mean inhibition was 67.6% (Fig. 1). These changes were significantly different (p < 0.01) from those observed in nontransgenic mice (Fig. 1).
TABLE 3. Catalase activity in brain regions ofSOD-transgenic mice and their nontransgmic littermates Cytosolic fraction Brain area' FCx
CPu Hip cer
19.21 f 3.64 52.39 f 2.75 50.26 f 8.53 48.28 f 6.24 42.1 1 ? 6.39
44.79 f 1.85' 96.66 f 4.9Y 97.32 f 4-89' 96.89 f 5.96d 73.98 f 3.47d
2.33 1.85 1.34 2.00 1.76
2.10 f 0.28 1.86 f 0.14 2.14 2 0.29 3.73 f 0.40 3.78 f 0.19
1.68 f 0.24 2.17 f 0.13 2.67 f 0.30 4.24 f 0.35 4.26 f 0.23
1.25 1.14 1.13
Catalase activity (U/mg of protein) was m d by a colorimetric procedure as described in Materials and Methods One unit of catalase activity was delined as 1 pmol of H D 2 redud/min. Data are mean 2 SEM values measured in duplicates in 12 SOD-transgenic and 12 nontransgenic mice. FCx, frontal cortex; CPu,caudate-putamen complex; Hip, hippocampus; Cer, cerebellum; Bs, brainstem. SOD-transgenic/nontransgenicactivity. p < 0.0I , "p < 0.05 compared with nontransgenic mice by tweway analysis of variance followed by Scheffe's test.
J. Neurmhem.. Vd.58. No. 5. 1992
S. PRZEDBORSKI E T AL.
TABLE 4. GSH-Px activitv in brain renions of SOD-transnenic mice and their nontranwenic littermates Cytosolic fraction
FCx CPU Hip Cer BS
228.55 f 19.26 238.48 f 16.54 274.02 & 11.72 271.84 f 14.93 318.43 f 11.81
237.57 f 22.04 248.57 & 13.35 244.51 f 12.18 269.28 f 14.66 297.09 ? 11.97
1.04 1.04 0.89 0.99 0.93
9.05 k 0.44 12.02 f 0.86 10.88 f 0.43 7.34 k 0.42 12.46 0.84
8.26 f 0.44 12.48 f 0.8 I 10.07 f 0.62 6.07 f 0.38 12.53 f 0.73
0.91 1.04 0.93 0.83 1.01
GSH-Px activity (U/mg of protein) was measured by a colorimetric procedure as described in Materials and Methods. One unit of GSH-Px activity was defined as 1 rmol of NADPH oxidized/min. Data are mean f SEM values measured in duplicates in 12 SOD-transgenic and 12 nontransgenic mice. Values were tested for differences by using a two-way analysis of variance followed by Scheffe's test. FCx, frontal cortex; CPu, caudate-putamen complex; Hip, hippocampus; Cer, cerebellum; BS, brainstem. SOD-transgenic/nontransgenic activity.
DISCUSSION The present study confirms the reports of increased CuZn-SOD activity in the brains of SOD-transgenic mice in comparison with their nontransgenic littermates (Epstein et al., 1987; Przedborski et al., 1991). The increase in CuZn-SOD is found in both the cytosolic and particulate fractions in all brain regions examined (Tables 1 and 2). The small, but significant, increase in particulate KCN-resistant SOD activity (see Table 2), which under our experimental conditions represents Mn-SOD (Bize et al., 1980), was unexpected (Hassan, 1988). Indeed, we had predicted that the activity of Mn-SOD might actually be decreased in transgenic mice in response to the increase in CuZn-SOD, in a fashion similar to the situation found in Down's syndrome blood cells (Sinet et al., 1975a; Baret et al., 1981; Sinet, 1982). Although the Mn-SOD and CuZnSOD are located on different chromosomes in normal situations in humans and mice (Bannister et al., 1987), it is possible that the human CuZn-SOD transgene might have incorporated itself in a position where it might have promoted the transcription of the Mn-SOD gene. Alternatively, posttranscriptional and posttranslational modification of the gene products might be
TABLE 5. SOD, catalase. and GSH-Px activities in the blood of SOD-transgenic mice and their nontransgenic littermates Enzymatic activity (U/ml of blood)
Total SOD Catalase GSH-PX
250.0 k 40.0 873.0 f 22.0 38.3 f 2.4
475.0 54.0b 916.0 38.0 39.2 f 1.8
Ratio" 1.90 1.05 1.02
Data are mean & SEM values measured in duplicates in three SOD-transgenic and three nontransgenic mice. a SOD-transgenic/nontransgenic activity. b p < 0.01 compared with nontransgenic mice by Student's twotailed t test. J. Neurochem., Val. 58, No. 5 , 1992
responsible for these increases in enzyme activity. Posttranslational modifications within the mitochondria are more likely because there were no differences in the enzyme activity within the cytosolic compartment between the two groups of mice (Table 2). It is of hrther interest that HeLa S3 cells, which are resistant to paraquat-induced oxidative stress, showed higher activities of both CuZn-SOD and Mn-SOD in particulate fractions (Krall et al., 1988), thus suggesting that both enzymes can be increased in tandem and also that particulate SOD activities might be of greater importance in providing protection against oxygen radical-induced toxicity. This contention is supported by the fact that mitochondria are important sites for the generation of oxygen-based radicals (Freeman and Crapo, 1982; Zhang et al., 1990). Under normal conditions, a delicate balance exists between the rate of H202formation via dismutation of 02-and its removal by catalase and GSH-Px (Freeman and Crapo, 1982). In the present study, we have found that SOD-transgenic mice showed catalase activity that was significantly higher than the value observed in the nontransgenic mice. We also found that the inhibition of brain catalase by injection of aminotriazole was significantly greater in SOD-transgenic mice in comparison with their nontransgenic littermates (Fig. 1). These findings provide confirming evidence for the possibility that greater amounts of H202 may be produced in the brains of the SOD-transgenic mice. Thus, the increase in catalase activity measured in these animals (Table 3) might be an adaptative response to the higher levels of H202generated by the augmented SOD activity. We failed, however, to demonstrate any similar changes in GSH-Fk in the SODtransgenic mice, whereas in Down's syndrome blood cells, the enhanced CuZn-SOD activity is accompanied by augmented GSH-Px activity (Sinet et al., 19756; Frischer et al., 1981; Kedziora et al., 1982) while catalase levels are normal (Pantelakis et al., 1970).Because the K , of GSH-Px is lower than that of catalase for H202(Cohen and Hochstein, 1963), elevated levels of H202are probably metabolized preferentially by GSHPx in vivo, and thus SOD-transgenic mice should have
SCA VENGER ENZYMES IN Cu/Zn-SOD TRANSGENIC MICE
SOD-TRANSGENIC MICE 7
FIG. 1. Effect of aminotriazole on brain catalase activity in SODtransgenic mice and their nontransgenic littermates.Arninotriazole (m) was injected intraperitoneally at the dose of 1 g/kg into SODtransgenic and nontransgenic mice; SOD-transgenic and nontransgenic controls were injected with saline (0).One hour after the injection,all animals were killed, and brain catalase activity was measured in the different regions as described in Materials and Methods. Data are mean SEM (bars) of control catalase activity measured in duplicates in three mice per group; the control values, expressed as U/mg of protein, are listed in Table 3. FCx, frontal cortex; CPu, caudate-putamen complex; Hip, hippocampus; Cer, cerebellum; BS, brainstem. The statistical significanceof differences was calculated by three-way analysis of variance followed by Scheffes test: ***p< 0.001 compared with saline-injected mice, ""p < 0.01 compared with nontransgenic aminotriazole-injected mice.
demonstrated a compensatory increase in GSH-Px activity. Nevertheless, a similar lack of increase in GSHPx activity was observed in the brains of Down's syndrome fetuses that showed increased CuZn-SOD activity (Brooksbank and Balazs, 1984). These results suggest that catalase may be more inducible than GSHPx in the presence of high levels of H202.This postulate is supported by previous studies, using isolated hepatocytes, indicating that mainly catalase activity was increased in concert with increased production of H202 (Jones et al., 1981; Jones, 1982) and that, at higher H202concentrations, catalase has a greater affinity toward H202than GSH-Px (Chance et al., 1979). The recent report that CuZn-SOD and catalase are located within the same peroxisomal compartments (Keller et al., 1991) and our findings of a positive correlation between CuZn-SOD and catalase activities provide further support for the notion that H202generated by
SOD might depend more on catalase than on GSHPx for its breakdown to water. Nonetheless, the possibility that the human CuZn-SOD transgene might be acting as a promoter for catalase or might have an indirect effect on catalase activity also must to be considered. In summary, because of their potentiated arsenal of scavenging enzymes, SOD-transgenic mice may indeed be essential models for assessing the role of oxygenbased radicals in the pathogenesis of neurological disorders. Oxygen-based radicals, especially 02-,have been implicated in the pathogenesis of neuronal cell death following cerebral hypoxia, ischemia/reperfusion, hyperoxia, and trauma (Kontos, 1985; Chan, 1989; Hall and Braughler, 1989). It has been proposed that, during these different brain insults, free radicals may perturb antioxidant enzymes; as a consequence there may be secondary overproduction of free radicals with associated peroxidative damage to cell membranes (Kontos, 1985; Chan, 1989; Hall and Braughler, 1989; Siesjo et al., 1989). Although increased lipid peroxidation has been clearly documented in several of these conditions, consistent changes in the activities of antioxidant enzymes have not been demonstrated. Specifically, several studies have reported changes in antioxidant enzymes during brain hypoxia and/or ischemia (Chan et al., 1988;Agardh et al., 1991;Sutherland et al., 1991), whereas others have failed to find any significant modifications during ischemia or reperfusion (Mishra and Delivoria-Papadopoulos, 1988; Michowiz et al., 1990; Mishra et al., 1990). Nevertheless, increasing brain SOD levels by administration of polyethylene glycol-conjugated (Liu et al., 1989) or of liposome-entrapped (Imaizumi et al., 1990)CuZn-SOD was reported to cause reduction of the extent of cortical infarction following focal brain ischemia. Similarly, ischemia (Kinouchi et al., 1990) and cold-induced trauma (Chan et al., 1991)caused lessdamage in SODtransgenic mice compared with their nontransgenic littermates. It is of further interest that glutamate neurotoxicity is also significantly reduced in SOD-transgenic neurons that contained a 2.5-fold increase in CuZn-SOD activity (Chan et al., 1990). Finally, because excitatory amino acid neurotoxicity has been implicated in neuronal cell death induced by ischemia (Pellegrini-Giampietroet al., 1988)as well as N-methyl4-phenyl-172,3,6-tetrahydropyridine (Turski et al., 1991) and because free radicals may be involved in excitotoxicity (Pellegrini-Giampietro et al., 1988, 1990), these data raise the intriguing possibility that excitatory amino acid and oxygen-based radical toxicity may interact to cause the appearance of several neurodegenerative disorders. This statement entails a combined approach using antiexcitotoxins and free radical scavengers in the treatment of some of these diseases (Choi, 1990).
Acknowledgment: This work was supported by the National Fund for ScientificResearch (Belgium),the Parkinson Disease Foundation (New York, NY, U.S.A.), the Fulbright FounJ. Neurochem., Vol. 58, No. 5, 1992
S. PRZEDBORSKI ET AL.
dation, and the National Institute of Mental Health (grant R29 MH 47509) and in part by the National Institute of Aging (grant AG-08958). T h e authors also wish to thank the anonymous reviewers for their thoughtful comments and suggestions.
REFERENCES Aebi H. (1984)Catalase in vitro. Methods Enzymol. 105, 121-126. Agardh C.-D., Zhang H., Smith M.-L., and Siesjo B. K. (1991)Free radical production and ischemic brain damage: influence of postischemic oxygen tension. Int. J. Dev. Neurosci. 9, 127-138. Badwey J. A. and Karnovsky M. L. (1980)Active oxygen species and the functions of phagoqtic leukocytes. Annu. Rev. Biochem. 49,695-126. Bannister J. V., Bannister W. H., and Rotilio G. (1987)Aspecfs of the Structure, Function, and Applications of Superoxide Dismutase, pp. 11 1-179. CRC Press, Boca Raton, Florida. Baret A., Baeteman M. A., Mattei J. F., Michel P., Broussolle B., and Giraud F. (1981)Immunoreactive Cu SOD and Mn SOD in the circulating blood cells from normal and trisomy 21 subjects. Biochem. Biophys. Res. Commun. 98, 1035-1043. Bize I. B., Oberley L. W., and Moms H. P. (1980)Superoxide dismutase and superoxide radical in Moms hepatomas. Cancer Res. 40,3686-3693. Brooksbank B. W. and Balazs R. (1984)Superoxide dismutase, glutathione peroxidase and lipoperoxidation in Down’s syndrome fetal brain. Dev. Brain Res. 16, 31-44. Cadet J. L. (1988)Oxyradical mechanisms in the central nervous system: an overview. Int. J. Neurosci. 40, 13-18. Cadet J. L., Lohr J. B., and Jeste D. V. (1986)Free radicals and tardive dyskinesia. Trends Neurosci. 9, 108-109. Cadet J. L., Przedborski S., Kostic V., Jackson-Lewis V., Carlson E., and Epstein C. J. (1990)Quantitative autoradiographic distribution of [3H]mazindol-labeled dopamine uptake sites in the brains of superoxide dismutase transgenic mice. Brain Res. Bull. 25, 187-192. Chan P. H. (1989)The role of oxygen radicals in brain injury and edema, in Cellular Antioxidant Defence Mechanisms (Chow C. K., ed), pp. 89-109.CRC Press, Boca Raton, Florida. Chan P. H., Chu L., and Fishman R. A. (1988)Reduction of activities of superoxide dismutase but not of glutathione peroxidase in rat brain regions following decapitation ischemia. Brain Res. 439, 388-390. Chan P. H., Chu L., Chen S . F., Carlson E. J., and Epstein C. J. (1990) Reduced glutamate neurotoxicity in transgenic mice overexpressing human CuZn-superoxide dismutase. Stroke 21, Ill-80-111-83. Chan P. H., Yang G. Y., Chen S. F., Carlson E., and Epstein C. J. ( 1991) Cold-induced brain edema and infarction are reduced in transgenic mice overexpressing CuZn-superoxide dismutase. Ann. Neurol. 29,482-486. Chance B., Sies H., and Boveris A. (1979)Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-605. Choi D. W. (1990)Cerebral hypoxia: some new approach and unanswered questions. J. Neurosci. 10, 2493-2501. Cohen G. (1984)Oxy-radical toxicity in catecholamine neurons. Neurotoxicology 517-82. Cohen G. and Hochstein P. (1963)Glutathione peroxidase: the primary agent for the elimination of H202in erythrocytes. Biochemistry 2, 1420-1428. Cross C. E., Watanabe T. T., Hasegawa G. K., Goralnik G. N., Roertgen K. E., Kaizu T., Reiser K. M., Gorin A. B., and Last J. A. (1979)Biochemical assays in lung homogenates: artifacts caused by trapped blood after perfusion. Toxicol. Appl. Pharmacol. 48,99-109. Dexter D. T.,Carter C. J., Wells F. R., Javoy-Agid F., Agid Y., Lees A., Jenner P., and Marsden C. D. (1989)Basal lipid peroxidation in substantia nigra is increased in Parkinson’s disease. J. Neurochem. 52,381-389. Epstein C. J., Avraham K. B., Lovett M., Smith S., Elroy-Stein O., Rotman G., Bry C., and Groner Y.(1987)Transgenic mice with J. Neurochem., Vol. 58, No. 5. 1992
increased Cu/Zn-superoxide dismutase activity: animal model of dosage effects in Down syndrome. Proc. Natl. Acad. Sci. USA 84,8044-8048. Flohe L. and Gunzler W. A. (1984)Assays ofglutathione peroxidase. Methods Enzymol. 105, I 14-121. Freeman B. A. and Crapo J. D. (1982)Biology ofdisease. Free radicals and tissue injury. Lab. Invest. 47,412-426. Fridovich I. (1986)Superoxide dismutases. Adv. Enzymol. 58, 6191. Fried R. (1975)Enzymatic and non-enzymatic assay of superoxide dismutase. Biochimie 57,657-660. Frischer H., Chu L. K., Ahmad T., Justice P., and Smith G. F. (1981) Superoxide dismutase and glutathione peroxidase abnormalities in erythrocytes and lymphoid cells in Down’s syndrome. Prog. Clin. Biol. Res. 55,269-283. Gaunt G. and DeDuve C. (1 976)Subcellular distribution of Damino acid oxidase and catalase in rat brain. J. Neurochem. 26, 749759. Green F. 0.and Feinstein R. N. (1957)Quantitative estimation of 3-amino-l,2,4-triazole. Anal. Chem. 29, 1658-1660. Hall E. D. and Braughler J. M. (1989)Central nervous system trauma and stroke. 11. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic. Biol. Med. 6, 303-313. Halliwell B. and Gutteridge J. M. (1985~) Oxygen radicals and the nervous system. Trends Neurosci. 5, 22-26. Halliwell B. and Gutteridge J. M. (1985b)The importance of free radicals and catalytic metal ions in human diseases. Mol. Aspects Med. 8,89-193. Hassan M. H. (1988)Biosynthesis and regulation of superoxide dismutases. Free Radic. Biol. Med. 5, 317-385. Imaizumi S.,Woolworth V., Fishman R. A., and Chan P. H. (1990) Liposome-entrapped superoxide dismutase reduces cerebral infarction in focal cerebral ischemia. Stroke 21, 13 12-13 17. Jones D. P. (1982)Intracellular catalase function: analysis of the catalatic activity by product formation in isolated liver cells. Arch. Biochem. Biophys. 214,806-814. Jones D. P., Eklow L., Thor H., and Orrenius S. (1981)Metabolism of hydrogen peroxide in isolated hepatocytes: relative contributions of catalase and glutathione peroxidase in decomposition of endogenously generated H202.Arch. Biochem. Biophys. 210, 505-5 16. Kedziora J., Lukaszewicz R., Koter R., Bartosz G., Pawlowska B., and Aitkin D. (1982)Red blood cell glutathione peroxidase in simple trisomy 21 and translocation 21/22.Experientia 38, 543544. Keller G.-A., Warner T. G., Steimer K. S., and Hallewell R. A. ( 199 1) CuZn superoxide dismutase is a peroxisomal enzyme in human fibroblasts and hepatoma cells. Proc. Natl. Acad. Sci. USA 88, 7381-7385. Kinouchi H., Imaizumi S., Carlson E., Epstein C. J., and Chan P. H. (1990)Focal cerebral ischemic infarction and brain edema are reduced in transgenic mice overexpressing human superoxide dismutase. (Abstr) Soc. Neurosci. Abstr. 20, 276. Kontos H. A. (1985)Oxygen radicals in cerebral vascular injury. Circ. Res. 57, 508-5 16. Krall J., Bagley A. C., Mullenbach G. T., Hallewell R. A., and Lynch R. E. (1988)Superoxide mediates the toxicity of paraquat for cultured mammalian cells. J. Biol. Chem. 263, 1910-1914. Ledig M., Fried R., Ziessel M., and Mandel P. (1982)Regional distribution of superoxide dismutase in rat brain during postnatal development. Dev. Brain Res. 4, 333-337. Liu T. H.. Beckman J. S., Freeman R. A., Hogan E. L., and Hsu C. Y. (1989)Polyethylene glycol-conjugated superoxide dismutase and catalase reduce ischemic braininjury. Am. J. Physiol. 256. H589-H593. Lowry 0. H., Rosebrough N. J., Farr A. L., and Randall R. J. (I95I) Protein measurement with the Fohn phenol reagent. J. Biol. Chem. 193,265-275. Maral J., Puget K., and Michelson A. M. (1977)Comparative study of superoxide dismutase, catalase and glutathione peroxidase levels in erythrocytes of different animals. Biochem. Biophys. Res. Commun. 77, 1525-1535.
SCA VENGER ENZYMES IN Cu/Zn-SOD TRANSGENIC MICE Michowiz S. D., Melamed E., Pikarsky E., and Rappaport Z. H. (1 990) Effect of ischemia induced by middle cerebral artery occlusion on superoxide dismutase activity in rat brain. Stroke 21, 161 3-1617. Mishra 0. P. and Delivoria-Papadopoulos M. (1988) Anti-oxidant enzymes in fetal guinea pig brain during development and the effect of maternal hypoxia. Dev. Brain Res. 42, 173-179. Mishra 0. P., Delivoria-PapadopoulosM., and Wagerle L. C. (1990) Anti-oxidant enzymes in the brain of newborn piglets during ischemia followed by reperfusion. Neuroscience 35,2 1 1-2 15. Noh1 H. and Hegner D. (1978) Evidence for the existence of catalase in the matrix space of rat-heart mitochondria. FEBS Lett. 89, 126-1 30. Pantelakis S. N., Karaklis A. G., Alexiou D., Vardas E., and Valaes T. ( 1970) Red cell enzymes in trisomy 2 1. Am. J. Hum. Genet. 22, 184-193. Pellegrini-Giampietro D. E., Cherici G., Alesiani M., Carla V., and Moroni F. (1988) Excitatory amino acid release from rat h i p pocampal slices as a consequence of free radical formation. J. Neurochem. 51, 1960-1963. Pellegrini-Giampietro D. E., Cherici G., Alesiani M., Carla V., and Moroni F. ( 1990) Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J. Neurosci. 10, 1035-1041. Perumal A. S., Tordzro W. K., Katz M., Jackson-Lewis V., Cooper T. B., Fahn S., and Cadet J. L. (1989) Regional effects of 6hydroxydopamine (6-OHDA) on free radical scavengers in rat brain. Brain Res. 504, 139-141. Przedborski S., Kostic V., Jackson-Lewis V., Carlson E., Epstein C. J., and Cadet J. L. (1990) Transgenic mice expressing the human SOD gene are resistant to MPTP-induced toxicity. (Abstr) SOC.Neurosci. Abstr. 16, 1260. Przedborski S., Kostic V., Jackson-Lewis V., Carlson E., Epstein C. J., and Cadet J. L. (199 1) Quantitative autoradiographic distribution of [3H]-MPTPbinding in the brains of superoxide dismutase transgenic mice. Brain Res. Bull. 26, 987-99 1.
Saggu H., Cooksey J., Dexter D., Wells F. R., Lees A., Jenner P., and Marsden C. D. (1989) A selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra. J. Neurochem. 53,692-697. Siesjo B. K., Agardh C.-D., and Bengtsson F. (1989) Free radicals and brain damage. Cereb. Brain Metab. Rev. 1, 165-21 1. Sinet P. M. (1982) Metabolism of oxygen derivatives in Down’s syndrome. Ann. NY Acad. Sci. 396, 83-94. Sinet P. M., Lavelle F., Michelson A. M., and Jerome H. (1975~) Superoxide dismutase activities of blood platelets in trisomy 2 1. Biochem. Biophys. Res. Commun. 67,904-909. Sinet P. M., Michelson A. M., Bazin A., Lejeune J., and Jerome H. (19756)Increase in glutathione peroxidax activity in erythrocytes from trisomy 21 subjects. Biochem. Biophys. Res. Comrnun. 67, 910-91 5. Sinet P. M., Heikkila R. E., and Cohen G. (1980) Hydrogen peroxide production by rat brain in vivo. J. Neurochem. 34, 142 1 - 1428. Slot J. W., Geuze H. J., Freeman B. A., and Crapo J. D. (1986) Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parenchymal cells. Lab. Invest. 55,363-371. Sutherland G., Bose R., Louw D., and Pinsky C. (1991) Global elevation of brain superoxide dismutase activity following forebrain ischemia in rat. Neurosci. Lett. 128, 169-172. Turski L., Bressler K., Rettig K.J., Loschmann P.-A., and Wachtel H. (1991) Protection of substantia nigra from MPP+ neurotoxicity by N-methyl-Baspartateantagonists. Nature 349,414-41 8. Weisiger R. A. and Fridovich I. (1973) Mitochondria1 superoxide dismutases. Site of synthesis and intramitochondrial localization. J. Biol. Chem. 248,4793-4796. Yusa T., Beckman J. S., Crapo J. D., and Freeman B. A. (1987) Hyperoxia increases H202production by brain in vivo. J. Appl. Physiol. 63, 353-358. Zhang Y., Marcillat O., Giulivi C., Emster L., and Davies K. J. ( 1990) The oxidative inactivation of mitochondria1 electron transport chain components and ATPase. J. Biol. Chem. 265, 1633016336.
J. Neurochem.. Vol. 58, No. 5, 1992