ROLE OF METALLOTHIONEIN AND OTHER ANTIOXIDANTS IN SCAVENGING SUPEROXIDE RADICALS AND THEIR POSSIBLE ROLE IN NEUROPROTECTION *

Pergamon

0197-0186 (95)O011+X

Neurochem.Irrt,Vol. 29, No, 2, pp. 145–152,1996 Publishedby ElsevierScienceLtd Printedin Great Britain.All riglrtsreserved 0197-0186/96315.00+0,00

ROLE OF METALLOTHIONEIN AND OTHER ANTIOXIDANTS IN SCAVENGING SUPEROXIDE RADICALS AND THEIR POSSIBLE ROLE IN NEUROPROTECTION* SABER HUSSAIN,l WILLIAM SLIKKER Jr”* and SYED F. ALI~]-3 ‘NeurochemistryLaboratory, Divisionof Neurotoxicology,National Center for Toxicological Research/FDA, Jefferson,AR 72079,U.S.A. ‘Departments of Pharmacology& Toxicologyand 3Biochemistry& Molecular Biology,University of Arkansas for Medical Sciences,Little Rock, AR 72205,U.S.A. (Received28 July 1995; accepted20 September 1995) Abstract-Based on the inhibition of nitrite formation by generating superoxide from xanthine/xanthine oxidase (X/XO) reaction system, metallothionein (MT) and other sulfhydryl containing amino acids have been selected to test their abilities to scavenge superoxide radicals. Different concentrations of metallothionein and other sulfhydryl containing molecules e.g. cysteine, N-acetyl-cysteine and glutathione, were used to assess superoxide scavenging properties. Metallothionein scavenges superoxide radical in a dose-dependent manner with increasing concentrations as evidenced by the inhibition of nitrite formation. Similar abilities to scavenge superoxide radicals were shown by cysteine, N-acetyl-cysteine. Glutathione also scavenges superoxide radical in a dose-dependent manner. In uitro experimentsdemonstrated that

metallothioneinis superiorin scavengingsuperoxideradicals compared to other sulfhydrylmoleculessuch as cysteine,N-acetyl-cysteineand even glutathione.The data, further, suggestthat metallothionein-11has a 6-foldhighercapacityto scavengesuperoxideradicalthan metallothionein-I.In addition,metallothioneinlike protein was isolated from different regions of mouse brain treated with zinc. Brain metallothioneinIike protein inhibits nitrite formation as demonstrated by other scavengers; however, the extent of inhibition is different by this protein isolated from different brain regions. The present study suggests that metallothioneins and metallothionein-like proteins isolated from mouse brain act as neuroprotective agents by scavenging superoxide radicals. Published by Elsevier Science Ltd.

Oxygen free radical species are considered to be involved in many important biological reactions. Several harmful reactions such as membrane peroxidation, DNA degradation and destruction of endothelial cells have been attributed to oxygen derived free radicals (Brown et al., 1995; Vile and Tyrrell, 1995). Furthermore, oxygen free radical species produced deleterious effects in various diseases such as cancer, AIDS, chemical induced liver damage, aging, rheumatoid arthritis and several autoimmune *This is one of nine original manuscripts on the subject of “antioxidants” related to a workshop organized by Dr Joe Marwah, which took place in Hollywood,Florida, U.S.A. on 12November 1994.Dr J. Marwah (National Institutes of Health) and Dr M. Ebadi (University of Nebraska Medical School) acted as executiveeditors in the refereeingof these articles. t Author to whom all correspondenceshould be addressed at: Neurochemistry Laboratory, Division of Neurotoxicology, HFT-132, N.C.T.R./FDA, 3900 NCTR Drive, Jefferson,AR 72079-9502,U.S.A.

diseases (Cerutti, 1985; Halliwell and Cross, 1991; Jaeschke, 1995). Oxygen free radicals are also thought to be involved in neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (Schapira, 1995; Gotz et al., 1994). Specific cellular enzymatic defense mechanisms mediated by superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) have been suggested to protect from oxygen free radical species (Harris, 1992; Michiels et al., 1994; Hussain et al., 1995a). It has also been suggested that sulfhydryl containing agents participate in protecting against radical induced damage (Rhee et al., 1994). Recently, a growing interest has been focused on a role of metallothionein (MT) as a radical scavenger because of its high thiol content which is involved exclusively in the formation of diamagnetic metal-thiolate clusters (Sato and Bremner, 1993). Several studies have suggested that MT scavengers reactive oxygen species (Thornalley and Vasak, 1985; Hart et al., 1995) and also provides cellular protection

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against a variety of DNA-damage induced by chemicals or radiation (Hanada et al., 1991). MTs belong to the class of low molecular weight proteins (mol.wt 600W7000), unique in properties such as high cysteine content, no aromatic amino acids, heat stability and inducibility on exposure to metals (Hamer, 1986). The major physiological roles of MTs are to detoxify and sequester toxic heavy metals by their ability to bind firmly with sulfhydryl (-SH) groups (Ochi et al., 1988; Hussain et al., 1995b). In addition, MT serves as a source of Cu and Zn for Cu, Zn dependent enzymes and co-factors in cellular metabolic processes that are under tightly regulated conditions (see review Ebadi, 1991; Suzuki and Kuroda 1994). Two major inducible isoforms of MT are found in most vertebrate tissue and known as MT1 and MT-II (Hamer, 1986). Uchida et al. (1991) have discovered putative MT-III in brain which is noninducible. MT-III has been shown to prevent in vitro neuronal sprouting and development of neurofibrillary tangles, which are common pathological conditions of the cerebral cortex in Alzheimer’s disease (Uchida et al., 1991; Uchida, 1994); however, the mechanism of this inhibition and the physiological significance of MT-III are not clear. In view of MT’s proposed protective role as a free radical scavenger it may have implications in brain neurological diseases. The importance of free radicals in neurological disorders is becoming increasingly recognized and there is a growing interest in mechanisms of antioxidant protection, with potential relevance for therapeutic use (Maxwell, 1995; Williams, 1995). Therefore, MT which is a unique protein requires further studies to explore its antioxidant nature and its implications in neuroprotection. The present study was designed to evaluate the superoxide scavenging abilities of MT I, II (rabbit liver) and MT like protein isolated from mouse brain. In addition, abilities of -SH containing molecules such as glutathione (GSH), L-CYSteine (CYS) and N-acetylcysteine (NAC) in scavenging superoxide radicals were also examined based on the inhibition of nitrite formation from hydroxylamine in the presence 0~generated from xanthine and xanthine oxidase system

(x/xO).

EXPERIMENTAL PROCEDURES

Materials Superoxide dismutase, xanthine, xanthine Oxidaw hydroxylammonium chloride, sulfanilic acid, u-naphthylamine, sucrose, glutathione (GSH), 5-5-dithiobis [2-nitrobenzoic acid] (DTNB), metallothionein-I and metallothio-

nein-11 were purchased from Metallothionein was obtained A. Liberator (Biosystems, U.S.A.). All the other reagents

Sigma Chemical Company. as a generous gift from Dr F, Dupont Medical products used were of analytical grade.

Incubation of MT, CYS, NAC and GSH with superoxide generation system to analyze their superoxide scavenging properties Different concentrations of commercially available MT from rabbit liver, CYS, NAC and GSH were incubated with 1.49 ml of 65 mM potassium phosphate buffer, 0.1 ml and 1.5mM xanthine and 0.1 ml of 100mM hydroxylammonium chloride. The reaction was initiated by the addition of 0.25 ml of xanthine oxidase (30 pg) and incubated at 25°C for 20 min (total volume 2 ml). After incubation time, 0.5 ml of sulfanilic acid were added to 0.5 ml of incubation mixture and incubated at room temperature for 5 min followed by 0.5 ml of a-naphthylamine. The absorbance was recorded at 530 nm. The principle of this assay was described by Elstner and Haupel (1976) and modified by Pattichis et al. (1994) to measure SOD antioxidant catalytic activity. Because of the instability of superoxide indirect methods to measure SOD activity other assays have been described generating superoxide, from xanthine which converts to uric acid catalyzed by xanthine oxidase, Superoxide radical produced by the X/XO reaction subsequently reacts with hydroxylammonium chloride to produce nitrite. The nitrite, in turn, reacts with sulfanilic acid to produce a diazonium compound, which subsequently reacts with ci-naphthylamine to produce a red azo compound, whose absorbance can be measured at 530 nm. The presence of SOD inhibits the production of nitrite, as this enzyme scavenges the superoxide radical produced from xanthine. The extent of production of red azo compound will, therefore, be inversely proportional to the amount of SOD present. In a similar way disputation of -SH containing compounds can be evaluated by using this assay system. The presence of MT or -SH containing amino acids inhibits the production of nitrite indicating their disputation of superoxide, Extraction of A4T-like low molecular proteinfrom brain tissue Male C57BL/6N mice of 12 month of age were obtained from the NCTR breeding colony. Mice were treated with different concentrations of ZnCl,. After 24 h brains were removed and dissected into different regions; caudate nucleus, frontal cortex, hippocampus, cerebellum and brain stem. The MT like low molecular weight protein was extracted by the method described by Otsuka et aL (1988) with brief modifications. Each region of the brain was homogenized in ice-cold sucrose (0.32 M; 20Y0W.V)followed by sonication for 10 s on ice and centrifuged at 12,000 g for 30 min at 4°C. The supernatant collected was boiled for 3 min and centrifuged at the same speed (12,000 g) for 30 min at 4“C. This supernatant is referred to as heat stable protein fraction. TCA was added to this fraction for a final concentration of 2°/0 and kept on ice for 10 min. Supernatant referred as the TCA soluble fraction was obtained by centrifugation at 12,000g for 30 min. The TCA soluble fraction was used as a source of MT-like protein and incubated with X/XO as described above to examine the disputation of superoxide radical. The purity of MT was shown by SDS– PAGE electrophoresis (&18Y0 gradient gel). The heat stable TCA fraction was prepared as described above and was

Antioxidant properties of metallothionein reduced by incubation with 100 mM Tris–HCl buffer (pH 8,8) containing 60 mM DTE, 40 mM EDTA, 4% SDS and 25% glycerol. The samples were boiled for 3 min and subjected to electrophoresis followed by silver staining to visualize the separated protein bands. (Otsuka et a[., 1988). Metallothionein (Sigma) was used as standard.

concentrations of MT were incubated in X/XO system where superoxide was generated. The results show dose-dependent inhibition of nitrite formation. The presence of MT that inhibits nitrite production indi-

cates that MT scavenges the superoxide radical produced from xanthine. The extent of nitrite inhibition was augmented with the increasing concentration of MT in reaction mixture. The order of nitrite inhibition by MT was as follows: MT-II > MT-I > MT-I&II. MT II was shown to have a high capacity to scavenge superoxide radical. For instance, MT-II at 1 pM concentration inhibits 810/0 whereas MT-I at the same concentration inhibits only 13°/0 of nitrite formation.

Statistical analysis

Data were analyzed by statistical analysis using ANOVA followed by individual tests conducted using Duncan’s Multiple Range test, andp values of 0.05 or less were considered as significant. RESULTS Inhibition of nitrite formation

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by MT

However, mixed forms of MT consisting of MT-I and MT-II show very little ability for nitrite production compared to separate forms of MT-I and MT-II. The presence of MT in the X/XO reaction mixture inhibits the production of nitrite indicating their disputation of superoxide.

To test the antioxidant properties of different forms of MT, the X/XO system was used to generate superoxide radical that reacts with hydroxylammonium chloride to produce nitrite. The extent of nitrite formation is inversely proportional to the amount of antioxidant present in the reaction mixture. The percentage of inhibition of nitrite formation in the presence of antioxidants demonstrates disputation of superoxide radicals. Figure 1 shows the inhibition of nitrite formation by different forms of metallothionein obtained from Sigma (MT-I from rabbit liver Cd-6%, Zn 0.6V0; MT11from rabbit liver Cd-5.7% and Zn 0.8Yo). Different

Inhibition of nitrite formation

by CYS, NAC, GSH

Sulfhydryl containing molecules CYS, NAC and GSH were studied to compare their capacity to scavenge superoxide radical. Figure 2 illustrates inhibition of nitrite formation in the presence of GSH in a concentration-dependent manner. Maximum inhibition

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MetallothioneinConcentration(PM) Fig. 1. MT inhibits nitrite formation by scavenging superoxide (0~-) radical in uitro. Different concentrations of MT (Sigma, from rabbit liver) were incubated with 1.49 ml of 65 mM potassium phosphate buffer, 0.1 ml of 1.5 mM xanthine and 0.1 ml of 100 mM hydroxylammonium chloride. The reaction was initiated by the addition of 0.25 ml of xanthine oxidase (30 pg) and kept at 25°C for 20 min (total volume 2 ml). After incubation time 0.5 ml of sulfanilic acid were added to 0.5 ml of incubation mixture and incubated at room temperature for 5 min followed by 0.5 ml of c+naphthylamine. The absorbance was recorded at 530 nm. The percentage of nitrite inhibition was calculated and expressed as a percentage of inhibition verses yM concentration of MT. Values represent meant SEM of 34 experiments.

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Fig. 2. (A) L-Cysteine (L-CYS), N-acetyl cysteine (NAC) and glutathione (GSH) inhibit nitrite formation by scavenging superoxide (O;-) radical in vitro. (B) SOD inhibits nitrite formation by scavenging superoxide (Oj-) radical. Different concentrations of L-CYS, NAC, GSH and SOD were incubated with 1.49 ml of 65 mM potassium phosphate buffer, 0.1 ml of 1.5mM xanthine and 0.1 ml of 100mM hydroxylarmnonium chloride. The reaction was initiated by the addition of 0.25 ml of xanthine oxidase (30 pg) and kept at 25°C for 20 min (total volume 2 ml). After incubation, 0.5 ml of sulfanilic acid were added to 0.5 ml of incubation mixture and incubated at room temperature for 5 min followed by 0.5 ml of cr-naphthylamine. The absorbance was recorded at 530 nm. The percentage of nitrite inhibition was calculated and exuressed as a percentage of inhibition vs. PM concentrations. Values represent mean+ SEM of 34 experiments.

(55%) was observed at 200 KM and thereafter no increase in nitrite inhibition was observed.,, Cysteine shows dose-dependent inhibition of nitrite formation. At 100 pM the inhibition of nitrite formation was 15°/0, increasing to 97°/0 at 4 mM. The NAC has been shown to inhibit nitrite formation with increasing concentration. NAC inhibits 2.9°/0 at 10 ,uM that increased to 82Y0 at 7 mM. Inhibition of nitrite by NAC at 25 PM concentration was 6.50/. while cysteine at this concentration had no effect on nitrite inhibition. Overall, NAC exhibited more scavenging abilities for radicals as compared to cysteine at lower concentrations. As expected the non-sulfhydryl amino acid, L-histidine was a week inhibitor for nitrite formations compared to sulfhydryl containing amino acids (data not shown). Therefore, the order of superoxide scavenging ability was GSH > NAC > CYS. A4T-like proteins isolated from mouse brain separated by gradient polyacrylamide gel electrophoresis

MT-like low molecular weight protein with isolated from different regions of the brain as described in the

materials and methods. To evaluate the presence of lowmolecular weight protein, heat stable TCA soluble fraction from hippocampus separated on &18% gradient polyacrylamide electrophoresis (Fig. 3). Figure 3, lanes A, B and C represent several protein bands separated from hippocampus before heat treatment. In contrast, after heat treatment and solubilization in TCA, conspicuous low molecular weight ptoteins bands were observed (Fig. 3, Lane D,E,F) as evidenced by their migration close to MT (Fig. 3, Lane G) standard. Therefore, TCA soluble fractions contained low molecular weight proteins. Inhibition of nitrite formation by MT-like proteins isoIatedfrom different parts of the mouse brain

The TCA soluble fractions contained low molecular weight protein as evidenced by electrophoretic separation. Therefore, the same fractions were used in X/XO reaction system to evaluate their superoxide scavenging abilities. Inhibition of nitrite formation by MT-like low molecular weight proteins from control and zinc treated different regions of the mouse brain

Antioxidant properties of metallothionein

ABC

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DEFG

Fig. 3. Identification of MT-like protein from the hippocampus of mouse brain. Mice were injected with ZnC12and hippocampi were dissected and homogenized in ice-cold sucrose (0,32 M; 20% w/v) followed by sonication for 10 s on ice and centrifuged at 12,000g for 30 min at 4“C. The supernatant collected was boiled for 3 min and centrifuged at the same speed (12,000 g). TCA was added to this fraction for a final concentration of 20/0and kept on ice for 10 min then centrifuged at 12,000g for 30 min. It was reduced by incubation with electrophoretic sample buffer containing 60 mM DTE. Prior to electrophoresis, samples were boiled for 3 min and loaded on the gel (6180/. gradient SDSPAGE). Lanes A, B and C. Control, 1 and 5 mg/kg, ZnC12,i.p. (supernatant obtained after centrifugation at 12000g). Lanes D, E and F. Control, 1 and 5 mg/kg, ZnC12,i.p., (TCA soluble fractions). Lane G. Metallothionein standard (Sigma).

is shown in Fig. 4. The results showed that the extent of nitrite formation inhibition is different in different regions. The general increase in nitrite formation inhibition was examined in all regions of the brain. However, significant increments of nitrite formation inhibition were observed in caudate nucleus and hippocampus (Fig. 4). The results suggest that caudate nucleus and hippocampus displayed higher degrees of scavenging properties.

DISCUSSION

Superoxide scavenging properties of MT and other sulfhydryl molecules were evaluated in the X/XO system in which superoxide generated reacts with hydroxylammonium chloride following nitrite production. MT has been used in the present study to evaluate its antioxidant/neuroprotective properties and compared with other sulfhydryl containing amino acids. It has recentIy been suggested that MT can be

induced in response to oxidative stress and may exert a protective role in oxidativelyinjured cells ( for review see Sato and Bremner, 1993). The results presented in this study indicate that MT scavenges superoxide radicals in a dose dependent manner. MT-I and mixed form of MT I&II exhibited less capability to scavenge superoxide as radicals compared to MT-II. MT-like protein isolated from control and Zn-treated mouse brain scavenged superoxide as evidenced by inhibition of nitrite formation. The forms of MT have not yet been characterized but electrophoretic separation demonstrated that this protein migrates close to standard MT on the 6-18Y0gradient gel. The conspicuous band suggests that it may include all forms of MT (i.e. MT-I, MT-II and MT-III). Further studies are underway to identify the presence of the different MT forms with MT cDNA probes. In vitro studies provide evidence that MT possessed antioxidant activity (Thornalley and Vasak, 1985; Hart et al., 1995). Embryonic cells from transgenic mice deficient in MT-I&I1 genes were reported to be

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Fig. 4. MT-like low molecular weight motein isolated from different regions of mouse brain scaverwes su~eroxide (O;–) radicals. The he~t st~ble TCA fractions were prepare~ as described in Experimen~al Procedures. The TCA soluble fractions from different regions of the brain (CN: Caudate nucleus; FC: Frontal cortex; HIP: Hippacampus; CE: Cerebellum; BS: Brain stem) were used as a source of MT-like protein and incubated with X/XO system to examine the disputation of superoxide radicals on the basis of inhibition of nitrite formation. Experiments were done in triplicate and expressed with mean ~ SEM. Statistical analysis was conducted by ANOVA and P values are indicated on the top of the bars.

more sensitive to oxidative stress (Lazo et al., 1995). CHO cells resistant to metal ions also displayed cross resistance to hydrogen peroxide suggesting the possible involvement of MT in protecting against the deleterious effects elicited by hydrogen peroxide (Cantoni et al., 1994). Over the last few years it has been suggested that MT provides protection against a variety of DNA damaging chemicals and radiation (Hanada et al., 1991; Lohrer and Robson, 1989; Dudek et al., 1993). CYS and NAC are known to be involved as antioxidant in metabolic processes (De Vries and De Flora, 1993; Malorni et al., 1995); however, their abilities to scavenge superoxide radicals in the present study are relatively weaker as compared to MT. These results suggest that MT has a higher capability in scavenging superoxide radical as compared to other sulfhydryl containing molecules. The spectroscopic studies have established that all 20 cysteine residues out of a total of 61 amino acids in MT are reported to be involved in metal binding of seven Zn and Cd ions, giving rise to unique diamagnetic metal-thiolate clusters (Otvos and Armitage, 1980; Vasak and Kagi, 1981; Kagi and Schaffer, 1988). This special property of MT may make it unique in its nature to act as an antioxidant/neuroprotective agent. The primary mode of free radical attack of MT could be the -SH

groups, although details of the mode of action of MT as an antioxidant are not known. MT that forms clusters of thiolate bonds by coordination between -SH groups and metals might have been enhanced by its ability to scavenge superoxide compared to other -SH containing molecules. It is also possible that MT may have several binding sites to quench superoxide radicals. However, the scavenging potential of SOD is much greater than MT. It has been demonstrated that MT induction and increased SOD activity approximately paralleled the time-course of X-ray irradiation (Sato et al., 1995; Shiraishi et al., 1983). Since SOD also contains Cu and Zn, it appears that metal coordination with proteins may have a significant role in biological systems. MT has many possible roles in brain neurological disease; for instance, MT is depleted in cortical regions of individuals manifesting Alzheimer’s disease (Uchida et al., 1991), because many of the neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases are augmented by direct involvement of oxygen free radicals. Therefore, the antioxidant properties displayed by MT-like protein have obvious neuroprotective implications. Studies are underway to test this hypothesis that in viuo induction of MT plays a neuroprotective role in neurological diseases mediated by oxygen free radical species.

Antioxidant properties of metallothionein Ackrrow[edgenrerrts-This research was supported in part by an appointment to the Postgraduate Research Program (SH) at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Food and Drug Administration.

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