Neurochemical Research, Vol. 22, No. I, 1997. pp. 17-22

Metallothionein Inducers Protect Against l-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Neurotoxicity in Mice

Patricia Rojas1 and Camilo Rios1,2

(Accepted June II, 1996)

l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) is a drug that induces parkinsonism in hu-man and non-human primates. Free radicals are thought to be involved in its mechanism of action.Recently, the participation of metallothionein as scavenger of free radicals has been proposed. Inthis work, we studied the effect of metallothionein inducers in MPTP neurotoxic action. Maleswiss albino mice were pretreated either with cadmium (1 mg/kg) or dexamethasone (5 mg/kg),two well-known inducers of metallothionein synthesis, and 5 hours later with an MPTP adminis-tration (30 mg/kg). Treatment schedule was repeated daily for either 3 or 5 consecutive days. AHanimals were killed 7 days after the last administration, and striatal dopamine and homovanillicacid contents were analyzed as an end-point of MPTP neurotoxicity. Striatal dopamine content ofcadmium plus MPTP-treated animals (3-days) increased by 32%, and 48% (5-days) vs MPTP-alone animals. Dexamethasone plus MPTP-treated group also showed increased dopamine levels28% (3-days) and 43% (5-days). MPTP treatment reduced striatal metallothionein concentration(49% vs control animals). Dexamethasone and cadmium increased metallothionein concentrationsin MPTP-treated groups, by 77% and 82% respectively. Results suggest that metallothionein in-duction provide a significant resistance factor against the deleterious effect of MPTP.

KEY WORDS: l-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Parkinson's disease; metallothionein; cadmium;dexamethasone; striatum.

INTRODUCTION

Metallothionein (MT) is a low-molecular-weight, sul-fur-rich, inducible protein which exhibits high affinity forcadmium, zinc and other heavy metals (1). This protein isthought to be involved in regulating the metabolism ofphysiologically important trace metals, such as copper andzinc, detoxification of toxic metals, free radical scavengingand antioxidant role (2). The mammalian MT family is

1 Departamento de Neuroquimica, Institute Nacional de Neurologia yNeurocirugia, "Manuel Velasco Suarez", SS, insurgentes Sur No.3877, Mexico 14269, D.F.

2 Address reprint requests to: Camilo Rios, Departamento de Neuro-quimica, Institute Nacional de Neurologia y Neurocirugia, "ManuelVelasco Suarez", SS, Av. Insurgentes Sur No. 3877, Mexico 14269,D.F. Phone: (52 5) 606 4040; fax: (52 5) 606 4040.

grouped in four isoforms: MT-I and MT-II are expressedin many tissues; MT-III isoform is expressed in human andmouse brains (3,4), and MT-IV is expressed in stratifiedsquamous epithelia (5). MT in the rat brain was identified,and characterized its properties (6,7).

Although liver is the most responsive organ to MTinduction, MT is also induced in other organs includingbrain, by various inducers as heavy metals, glucocorti-coid hormones, acute stress, some organic chemicals,physical stress, and infection (4,8,9). Only MT-I andMT-II isoforms are induced by such agents. On the otherhand, it is well-known that I-methyl-4-phenyI-1,2,3,6-tetrahydropyridine (MPTP) when administered to non-human primates and mice produces hypokinesia andneuronal damage similar to that observed in idiopathicParkinson's disease (10).

170364-3190/97/0100-0017$ 12.50/0 85 1997 Plenum Publishing Corporation

18 Rojas and Rios

MPTP also produces depletion of striatal dopamineand cellular degeneration of the nigrostriatal pathway,mimicking that observed in Parkinson's disease (11).The neurotoxic effect of MPTP is assessed by the DAand HVA depletory action of the neurotoxin (10), MPTPis biotransformed to the l-methyl-4-phenylpyridiniumion (MPP+), its toxic metabolite, by type-B monoamineoxidase (12). MPP+ is then accumulated into dopami-nergic neurons by the high affinity dopamine uptake sys-tem (13). The neurotoxic mechanism of the MPTPmetabolite is unknown, but its toxicity has been relatedto MPP+ inhibition of site I mitochondrial respiration(14,15) and/or to oxidative stress induction (16).

In previous studies, we found enhanced lipid per-oxidation after MPP+ administration to mice (17,18), aprocess dependent of free radicals overproduction, andalso a depletion of trace metal content in the MPTPmodel (19). MT is a protein related both with oxidativestress and trace metals, therefore, the purpose of thepresent study was to examine if the effects of MT in-ducers (cadmium and dexamethasone) can modulateMPTP neurotoxicity. Cadmium (Cd) and dexamethasone(Dex) were selected for the present study because theyare both good inducers of MT and they are thought tomediate MT synthesis through different mechanisms(20-22).

EXPERIMENTAL PROCEDURE

Adult male swiss albino mice (25-30 g) NIH bred in-house wereused in all the experiments. Animals were fed with Purina chow (Pur-ina, Mexico) and drank water freely, MPTP hydrochloride was ob-tained from Research Biochemicals (Wayland, MA, U.S.A.), methanolwas purchased from J. T. Baker, Mexico, KH2PO4, CdCl2, and sucrosewere purchased from E. Merck, Mexico and all other reagents werefrom Sigma Chemical Co. (St. Louis, MO, U.S.A.).

MT Inducers Pretreatment and MPTP Administration, Male swissalbino mice were injected i.p, with one of the MT inducers: Cd (1mg/kg) or Dex (5 mg/kg) and allocated for MPTP intoxication (30mg/Kg, i.p.) into one of the following groups of mice; group 1, controlanimals treated with saline solution; group II, animals injected withCd; group III, animals injected with Dex; group IV, animals treatedwith saline solution and 5 hours later with MPTP; group V, animalsinjected with Dex and 5 hours later with MPTP; group VI, animalstreated with Cd and 5 hours later with MPTP. Treatment schedule wasrepeated daily for either 3 or 5 consecutive days. All animals werekilled by dislocation 7 days after the last administration and the striataldopamine (DA) and homovanillic acid (HVA) contents were measuredby HPLC system.

Determination of DA and HVA, We analyzed striatal DA andHVA concentrations. Mice brains were removed quickly and their cor-pora striata were dissected out upon ice as described by Glowinskiand Iversen (23). An aliquot (500 ul) of antioxidant solution contain-ing 0.1% w/v Na2S2O5 in 0.05 M perchloric acid was added to thetissue and sonicated with a Lab-line ultratip Labsonic system (Lab-

line instruments, Melrose Park, IL). Samples were ccntrifuged at 4,000g for 10 min and the supernatants were kept at — 70°C until analyzed.

Striatal contents of DA and HVA were analyzed using HPLC-system (LC 250 Perkin-Elmer) with electrochemical detector (Me-trohm 656) and Hewlett-Packard 3396-11 integrator as previouslydescribed (19).

Calibration curves were constructed for DA and HVA. Sampleconcentrations were obtained by interpolation in the respective stan-dard curve. An Alltech Associates, Inc. (Deerrield, IL), adsorbosphorecatecholamine analytical column of 100 x 4.8 mm with 3 um particlediameter was used. The mobile phase consisted of aqueous phosphatebuffer (pII 3.2) which contained 0.2 mM sodium acetyl sulfate, 0.1mM EDTA, and 15% v/v of methanol. Water and methanol wereHPLC-grade reagents. Flow was 1.6 ml/mm. The detector potentialwas adjusted to 0.8 V vs. Ag/AgCl reference electrode. All sampleswere analyzed in duplicate.

Striatal Estimation of MT Proteins, The MT proteins concentra-tion of each tissue sample was estimated by the silver-saturationmethod (24). This method measures all MT isoforms (MT 1, II, I I I ,and IV). Mice were killed by dislocation after MPTP treatment for 5consecutive days as described above. MT proteins were analyzed incorpus striatum, as follows: all laboratory glassware, polypropylenetubes and disposable micropipettc tips were immersed for severalhours in 5% v/v concentrated HNO1/H2O thoroughly rinsed in deion-ized water and nitrogen gas dried before use, to avoid any possiblecontamination. Stainless steel dissection material was cleaned with arapid immersion in 3% v/v nitric acid and thoroughly rinsed withdeionized water prior to its use. Tissue samples of corpus striatumwere homogenized in 500 ul of ice-cold 0.05 M phosphate buffer (pH7.0) containing 0.015 M NaCl and 0.145 KCI. Silver ion aqueoussolution (20 mg/L, 250 ul) was added to tissue extract (500 ul) tobind cytosolic proteins and other ligands including MT proteins. Rathemoglobin (200 ul) was added and samples boiled for 2 minutes toprecipitate silver excess. Only silver bound to MT proteins is left inthe supernatant after two repeated hemoglobin-boiling treatments. MTproteins concentration was calculated from silver concentration in thesupernatant as determined by atomic absorption analysis using a Per-kin-Elmer 360 Atomic Absorption Spectrophotometer with HGA-2200graphite furnace. A deuterium arc background corrector was used tocompensate background signal. For both samples and standards, a 20ul aliquot of each final diluted solution was injected into the furnace.Furnace program was optimized by measuring the standards signalheight at several temperature settings, for both char and atomizationstages, according to the procedure of Welz (25). MT proteins detectionlimit was about 5 ug MT/g wet tissue.

Statistical Analysis, Data of DA and HVA values were statisti-cally analyzed using one-way analysis of variance (ANOVA), fol-lowed by Tukey's test. Results of MT proteins were analyzed byone-way analysis of variance (ANOVA) followed by Dunnet's test.Values of p < 0.05 and p < 0.01 were considered of statistical sig-nificance.

RESULTS

We observed no change in either weight gain, waternor food consumption in any treatment group.

Neither Cd nor Dex administrations during 3 or 5days changed dopamine content in striatum as comparedto values of respective control mice (Fig. 1 and 2). A

Metallothionein Inducers Protect Against MPTP Neurotoxicity 19

Fig, 1. Striatal dopamine content at 3 days of MPTP treatment. Resultsare expressed as mean ± SEM of n = 5 - 7 independent experi-ments.** Statistically different MPTP versus control, Cd and Dex p< 0.01, Tukey's test.* Statistically different MPTP versus MPTP +Cd and MPTP + dex, p < 0.05, Tukey's test. MPTP = l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Dex = Dexamethasone, Cd = Cad-mium.

Fig. 3. Striatal homovanillic acid content at 3 days of MPTP treatment.Results are expressed as mean ± SEM of n = 5 - 7 independentexperiments.* Statistically different MPTP versus control p < 0.01,Tukey's test. MPTP = l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine,Dex = Dexamethasone, Cd = Cadmium.

Fig. 2. Striatal dopamine content at 5 days of MPTP treatment. Resultsare expressed as mean ± SEM of n = 5 - 7 independent experi-ments.** Statistically different MPTP versus control, Cd and Dex p< 0.01, Tukey's test.* Statistically different MPTP versus MPTP +Cd and MPTP + Dex, p < 0.05, Tukey's test. MPTP = l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Dex = Dexamethasone, Cd = Cad-mium.

significant decrease in strtatal DA was observed in thegroup of mice with 3 days of MPTP treatment (Fig. I),as a result of the neurotoxic action of the compound.Administration of Cd plus MPTP to animals for 3 days,partially protected (32% compared to respective MPTPgroup) animals against the neurotoxic effect of MPTP(Fig. 1). The same treatment for 5 days increased DAcontent by 48% as compared with the respective MPTP-

Fig. 4. Striatal homovanillic acid content at 5 days of MPTP treatment.Results are expressed as mean ± SEM of n = 5 - 7 independentexperiments.** Statistically different from MPTP p < 0.05, Tukey'stest." Statistically different from the other groups MPTP. MPTP = 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Dex = Dexamethasone,Cd = Cadmium.

alone group (Fig. 2). Mice receiving Dexamethasone +MPTP as treatment showed significant protection bothin the 3 and 5 days of treatment groups, expressed asenhanced DA levels of 28% and 43%, respectively,against MPTP-treated mice values (Fig. 2).

The HVA content in the striatum of mice with 3days (Fig. 3) of MPTP treatment decreased significantly.No differences were found in the HVA levels of all othergroups. The HVA contents in the striata of mice with 5

20 Rojas and Rios

Fig. 5. Metailothionein concentrations (ug/g wet weight). Results areexpressed as mean ± SEM of n = 5 — 7 independent experiments,**Statistically different from control p < 0.01, Dunnet's test.* Statisti-cally different from control p < 0.05, Dunnet's test. MPTP = 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine, Dex = Dexamethasone,Cd = Cadmium.

days of treatment (Fig, 4), was found decreased signif-icantly in MPTP-alone group when compared with val-ues of control animals. Cd + MPTP and Dex + MPTPgroups showed increased striatal HVA levels (85% and65% respectively) as compared with respective MPTP-treated group values.

Mice exposed to cadmium showed a two-fold in-crease in their striatal MT proteins concentration (Fig.5). Mice exposed to Dex had also an approximately two-fold increase in their striatal MT proteins concentration(Fig. 5). In contrast, the levels of MT proteins were re-duced (49% vs control group values) in the striata ofMPTP-treated mice (see again Fig. 5).

Cd + MPTP treatment resulted in a significant in-crease in MT proteins content. A marked elevation (77%as compared to MPTP-alone values) of MT proteins con-centration was also observed in the striata of mice withDex + MPTP treatment.

DISCUSSION

The present study shows for the first time that Dexand Cd, both metallothionein inducers, protect partiallymice against MPTP neurotoxicity, measured as dopa-mine depletion. Even when DA depletion after MPTPwas not severe, it is interesting to note that the protectionof MT inducers was more important (in percentage) atthe highest MPTP dose used. The MT-inducers protec-tion to mice observed in the present study could be dueto the putative action of metallothionein as hydroxyl rad-

ical scavenger (26), as cytotoxic hydroxyl radicals areoverproduced in vivo by MPTP administration to ro-dents (27). It is interesting to remark that both Cd andDex administrations to mice enhanced MT striatal con-tent by a similar amount, and that these treatments pro-tected almost by the same extent against MPTPneurotoxicity, suggesting that protective effects of Cdand Dex are mediated by MT. Manganese, another MTsynthesis inducer, has also been reported to protectagainst MPTP/MPP+ neurotoxicity in mice (28). Bothin the present study and the previously reported one (28),protection afforded by MT synthesis inducers is partial,suggesting the involvement of non-MT mechanisms inMPTP neurotoxicity.

Recent observations suggest that MT might partic-ipate in intracellular defenses, as a response against re-active oxygen species overproduction in brain. It hasbeen reported that chemicals that produce oxidativestress can also induce the synthesis of MT (29).

MPTP is related to oxidative stress since its toxicmetabolite, MPP+, is able to increase lipid peroxidation(17,18), and to decrease copper and manganese in corpusstriatum after MPTP administration (19). It is possiblethat MT, that binds metals such as zinc (30) and copper,plays an important role in protecting dopaminergic neu-rons from free radicals induced by MPTP.

Oxidative stress is not only the result of reactiveoxygen species overproduction, it is also the conse-quence of decreased mechanisms of cells' protection(31). A constant finding in the literature is that MPTPadministration to mice produce a decrease in brain con-centrations of reduced glutathione (32), a well-knownthiol-dependent cellular mechanism of defense againstoxygen reactive species (31). In this regard, it is inter-esting to note that striatal MT content was also founddecreased in the present work, as a result of MPTP treat-ment to mice, suggesting decreased thiol-related de-fenses against free radicals, associated to MPTP action.This could explain why thiol-containing molecules pro-tect against MPTP neurotoxicity (33). 6-hydroxydopa-mine, another dopaminergic neurotoxin, also reducesMT concentration in the striatum (34), the same effectwe observed in this study for MPTP.

It has been proposed recently that the nucleophilicsulfhydryl groups in MT can interact with electrophilictoxins and, by regulating the intracellular redox poten-tial, may also act as an scavenger of free radicals (2). Adeficiency in MT-I and II genes produces enhanced sen-sitivity to oxidative stress (35), while overexpression ofMT genes confer cells resistance against potent oxidantmolecules, like nitric oxide (36). According to results ofthe present study, it is expected a low ability of MPTP-

Metallothionein Inducers Protect Against MPTP Neurotoxicity 21

treated mice to control reactive oxygen species overpro-duction.

In addition to reduce thiol-related defense mecha-nisms of cells, decreased striatal content of MT as aresult of MPTP action can explain in part the observeddepletion of striatal copper content produced by MPTPin mice (19), as this transition metal is thought to beregulated by MT.

The effect of MPTP on striatal MT content remainsto be explained, however, data presented here suggestthat MPTP interferes with MT synthesis, as both basaland induced (by Cd and Dex) MT contents (22) werereduced by MPTP treatment to mice.

Parkinson's disease has been associated with oxi-dative stress (37), reduced thiol-related defenses (38)and also to changes in trace metals concentrations (39),specially increased free iron (40). Both increased lipidperoxidation and copper depletion have also been re-ported in the substantia nigra of Parkinson's disease pa-tients post mortem (41,42). All these findings suggestthe existence of similar mechanisms of damage both inidiopathic Parkinson's disease patients and in MPTP-in-duced neurotoxicity, thus, results presented here suggestthat a possible alteration of MT in such patients couldbe expected.

ACKNOWLEDGMENTS

We thank to Norma Rojas and Gisela Cahero for their technicalassistance. We also thank to Dr. Arnulfo Albores for his technicalassistance to analyze MT. This study was supported in part byCONACyT grants 0044P-M9506, F256-M9207, and the Programa deApoyo a las Divisiones de Estudios de Posgrado 030324, UniversidadNacional Autonoma de Mexico.

REFERENCES

1.

2.

3.

4.

5.

6.

Kagi, J. H. R., and Schaffer, A. 1988. Biochemistry of metallo-thionein. Biochemistry. 27:8509-8515.Sato, M., and Bremner, I. 1993. Oxygen free radicals and metal-lothionein. Free Rad. Biol. Med. 14:325-337.Uchida, Y., Takio, K., Titani, K., Ihara, Y., and Tomonaga, M.1991. The growth inhibitory factor that is deficient in Alzheimer'sdisease is a 68 amino acid metallothionein-like protein. Neuron.7:337-347.Palmiter, R. D., Fidley, S. D., Whitmore, T. E., and Durnam, D.M. 1992. MT-III, a brain-specific member of the metallothioneinfamily. Proc. Natl. Acad. Sci. U.S.A. 89:6333-6337.Quaife, C. J., Findley, S. D., Erickson, J. C., Forelick, G. J., Kelly,E. J., Zambrowicz, B. P., and Palmiter, R. D. 1994. Induction ofa new metallothionein isoform (MT-IV) occurs during differenti-ation of stratified squamous epithelia. Biochemistry. 33:7250-7259.Ebadi, M. 1986. Characterization of a metallothionein-like proteinin rat brain. Biol. Trace Elem. Res. 11:101-116.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Ebadi, M., and Babin, D. 1989. The amino acid composition ofthe zinc-induced metallothionein isoforms in rat brain. Neuro-chem. Res. 14:69-73.Hidalgo, J., Borras, M., Garvey, J. S., and Armario, A. 1990.Liver, brain, and heart metallothionein induction by stress. J. Neu-rochem. 55:651-654.Choudhuri, S., McKim, J. M., and Klaassen, C. 1993. Differentialexpression of the metallothionein gene in liver and brain of miceand rats. Toxicol. Pharmacol. 119:1-10.Heikkila, R. E., Cabbat, F. S., Manzino, L., and Duvoisin, R. C.1984. Effects of MPTP on nigrostriatal dopamine in mice. Neu-ropharmacology 23:711-713.Gerlach, M., Riederer, P., Przuntek, H., and Youdim, M. B. H.1991. MPTP mechanisms of neurotoxicity and their implicationsfor Parkinson's disease. Eur. J. Pharmacol. Mol. Pharmacol. 208:273-286.Chiba, K., Trevor, A., and Castagnoli, N. 1984. Metabolism ofthe neurotoxic tertiary amine, MPTP, by brain monoamineoxidase. Biochem. Biophys. Res. Comtnun. 120:574-578.Chiba, K., Trevor, A. J., and Castagnoli, N. 1985. Active uptakeof MPP+, a metabolite of MPTP by brain synaptosomes.Biochem. Biophys. Res. Commun. 128:1228-1232.Nicklas, N. J., Vyas, I., and Heikkila, R. E. 1985. Inhibition ofNADH-linked oxidation in brain mitochondria by l-methyl-4-phenylpyridine, a metabolite of the neurotoxin,l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine. Life Sci. 36:2503-2508.Ramsay, R. R., Kowal, A. T., Johnson, M. K., Salach, J. I., andSinger, T. P. 1987. The inhibition site of MPP+, the neurotoxicbioactivation product of l-methyl-4-phenyl-l,2,3,6-tetrahydropyr-idine is near the Q-binding site of NADH dehydrogenase. Arch.Biochem. Biophys. 259:645-649.Adams, J. D., and Odunze, 1. N. 1991. Biochemical mechanismsof l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine toxicity. Couldoxidative stress be involved in the brain? Biochem. Pharmacol.41:1099-1105.Rios, C., and Tapia, R. 1987. Changes in lipid peroxidation in-duced by l-methyl-4-phenylpyridinium in mouse brain homogen-ates. Neurosci. Lett. 77:321-326.Rojas, P., and Rios, C. 1993. Increased striatal lipid peroxidationafter intracerebroventricular MPP+ administration to mice. Phar-macol. Toxicol. 72:364-368.Rios, C., Alvarez-Vega, R., and Rojas, P. 1995. Depletion of cop-per and manganese in brain after MPTP treatment of mice. Phar-macol. Toxicol. 76:348-352.Andrews, G. K. 1990. Regulation of metallothionein gene expres-sion. Prog. Food Nutr. Sci. 14:193-258.Liu, J., Liu, Y. P., Sendelbach, L. E., and Klaassen, C. D. 1991.Endotoxin induction of hepatic metallothionein is mediatedthrough cytokines. Toxicol. Appl. Pharmacol. 109:235-240.Zheng, H., Berman, N. E., Klaassen, C. D. 1995. Chemical mod-ulation of metallothionein I and III mRNA in mouse brain. Neu-rochem. Int. 27:43-58.Glowinski, J., and Iversen, L. L. 1966. Regional studies of cate-cholamines in the rat brain. Disposition of 3H-norepinephrine, 3H-dopamine, and 3H-DOPA in various regions of the brain. J.Neurochem. 13:655-669.Scheuhammer, A. M., and Cherian, G. M. 1986. Quantification ofmethallothionein by silver-saturation method. Toxicol. Appl. Phar-macol. 82:417^125.Welz, B. 1976. Atomic spectroscopy. Verlag-Chemie, Weinheim,New York.Thornalley, P. J., and Vasak, K. 1985. Possible role for metallo-thionein in protection against radiation-induced oxidative stress.Kinetics and mechanism of its reaction with superoxide and hy-droxyl radicals. Biochim. Biophys. Acta. 827:36—47.Wu, R., Chiueh, C. C., Pert, A., and Murphy, D. L. 1993. Ap-parent antioxidant effect of 1-deprenyl on hydroxyl radical for-

22 Rojas and Rios

28.

29.

30.

31.

32.

33.

34.

35.

mation and nigral injury elicited by MPP+ in vivo. Eur. J.Pharmacol. 243:241-247.Rojas, P., and Rios, C. 1995. Short-term manganese pretreatmentpartially protects against l-methyl-4-phenyl-l,2,3,6-tetrahydropyr-idine neurotoxicity. Neurochem. Res. 20:1217—1223.Bauman, 3, W,, Liu, )., Liu, P., and Klaassen, C. D. 1991. Increasein metallothionein production by chemicals that induce oxidativestress. Toxicol. Appl. Pharmacol. 110:347-354.Ebadi, M., Iversen, P. L, Hao, R., Cerutis, D. R., Rojas, P.,Happe, H. K., Murrin, L. C., and Pfeiffer, R. F. 1995. Expressionand regulation of brain metallothionein. Neurochem. Int. 27:1-22.Halliwell, B,, and Gutteridge, J. M. C. 1985. Free radicals in bi-ology and medicine, Oxford-Claredon.Ferraro, T. N., Golden, G. T., DeMattei, M., Hare, T. A., andFariello, R. G. 1986. Effect of l-methyl-4-phenyl-2,3,6-tetrahy-dropyridine (MPTP) on glutathione in the extrapyramidal systemof the mouse. Neuropharmacology. 25:1071-1074.Oishi, T., Hasegawa, E,, and Murai, Y. 1993. Sulfhydryl drugsreduce neurotoxicity of l-methyl-4-phenyl-l,2,3,6-tetrahydropyr-idine (MPTP) in the mouse. J. Neural Transm. (P-D Sect). 6:45-52,Shiraga, H., Pfeiffer, R. F., and Ebadi, M. 1993. The effects of 6-hydroxydopamine and oxidative stress on the level of brain me-tallothionein. Neurochem. Int. 23:561-566,Lazo, J. S., Kondo, Y., Dellapiazza, D., Michalska, A. E., Chook,H. A., and Pitt, B. R. 1995. Enhanced sensitivity to oxidativestress in cultured embryonic cells from transgenic mice deficientin metallothionein I & II genes. J. Biol. Chem. 270:5506-55)0.

36.

37.

38.

39.

40.

41.

42.

Schwarz, M. A., Lazo, J. S., Yalowich, J. C., Allen, W. P., Whit-more, M., Bcrgonia, H. A., Tzeng, E., Billiar, T. R., Robbins, P.D., Lancaster Jr., J. R., and Pitt, B. R. 1995. Metallothionein pro-tects against cytotoxic and DNA-damaging effects of nitric oxide,Proc. Natl. Acad. Sci. USA. 92:4452-4456.Halliwel, B. 1989. Oxidants and the central nervous system: somefundamental questions. Is oxidant damage relevant to Parkinson'sdisease, Alzheimer's disease, traumatic injury or stroke? ActaNeurol. Scand. 126:23-33.Perry, T. L., Godin, D. V., and Hasan, S, 1982. Parkinson's dis-ease: a disorder due to nigral glutathione deficiency? Neurosci.Lett. 33:305-310.Dexter, D. T., Carayon, A., Javoy-Agid, F., Agid, Y., Wells, F.R., Daniels, S. E., Lees, A. J., Jenner, P., and Marsden, C. D.1991. Alterations in the levels of iron, ferritin and other tracemetals in Parkinson's disease and the other neurodegenerative dis-ease affecting the basal ganglia. Brain. 114:1953-1975.Gerlach, M., Ben-Shachar, D., Riederer, P., and Youdim, M. B.H. 1994, Altered brain metabolism of iron as a cause of neurod-egenerative diseases? J. Neurochem. 63:793-807.Dexter, D. T., Carter, C. J., and Wells, F. R. 1989. Basal lipidperoxidation in substantia nigra is increased in Parkinson's dis-ease. J. Neurochem. 52:381-389.Riederer, P., Sofic, E., Rausch, W. D., Schmidt, B., Reynolds, G.P., Jellinger, K., and Youdim, M. B. H. 1989. Transition metals,ferritin, glutathione, and ascorbic acid in parkinsonian brains. J.Neurochem. 52:515-520.