PII: 80278-5846(99)00077-9

CHABGES OF METALLOTHIONEIN I + II PROTEINS IN THE BRAIN AFTER l-METHYL-4-PHENYLPYRIDINIUM ADMINISTRATION IN MIC$

PATRICIA ROJAS’ , JUAN HIDALG02, MANUCHAIR EBAD13 and CAMILO RIO!?

‘Laboratorio de Neurotoxicologia, lnstituto National de Neurologia y Neurocirugia, Mexico D.F., Mexico, ‘Departamento de Biologia Celular y Fisiologia, Unidad de Fisiologia Animal, Facultad de Ciencias. Universidad Autonoma de Barcelona, Barcelona, Spain, 3Department of Pharmacology, University of

Nebraska Medical Center Omaha, Nebraska, USA. 4Departamento de Neuroquimica. lnstituto National de Neurologia y Neurocirugia, Mexico City, Mexico.

(Final form, November 1999)

Abstract

Rojas, Patricia, Juan Hidalgo, Manuchair Ebadi. and Camilo Rios: Evaluation of metallothionein I+11 proteins after 1-methyWphenylpyridinium administration. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 2ggg, a,

pp. 143-154.01999 Eleevier Science Inc.

1. 1-methyl-4-phenylpyridinium (MPP+) is a drug that induces a Parkinson’s_like syndrome in several species. Oxidative stress resulting from either excess generation or reduced scavenging of free radicals has been proposed to play a role in its neurotoxicity.

2. It has been suggested that metallothionein (MT) protects against oxidative damage of the central nervous system produced by overproduction of free radicals.

3. This study examined the effect of MPP+ on metallothionein I+11 protein content in different brain regions,.

4. NIH mice were injected with MPP+ (4.5,g.O or 18 a/ 3 u) into their right lateral ventricle. 5. Corpus striatum, cerebellum, midbrain, frontal cortex and hippocampus were dissected out and their

metallothionein concentrations were analyzed by radioimmunoassay. 6. MPP+ reduced the concentration of MT l+II proteins (38%) only in the striatum. 7. The results suggest that changes in MT I+11 content may be associated with MPP+ neurotoxicity.

Kevwords: free radicals, MPP+, metallothionein, oxidative stress, Parkinson’s disease.

” Abbrevlatms. metallothionein (MT), I-methyl-4-phenylpyridinium (MPP+). 1-methyl-4-phenyC1.2.3.6- tetrahydropyridine (MPTP).

143

144 P. Rojas et al.

Introductim

Metallothionein (MT) isoforms are low molecular weight, metal- and sulfur-rich proteins with high cysteine

content and no aromatic acids, histidine or disulfide bonds (Klgi, 1993). The mammalian MT family consists

of four major isoforms: MT-I and MT-II are expressed in various organs and their synthesis is regulated by

many factors including metals, glucocorticoids, endotoxin. cytokines, stress, and radiation (Kagi, 1993). MT-

Ill is prevailing in zinc-containing neurons, and it is thought to play a role in the uptake of zinc into neurons

and the transport of the metal within neurons and their synaptic vesicles (Masters et al., 1994). MT-IV is

restricted to stratified squamous epithelium (Quaife et al., 1994). MT isoforms are found also in glia (Young,

et al., 1991).

MT is the most abundant and important thiol source in the brain. In this organ, MT tsoforms have been

proposed to participate in the transport, accumulation, and compartmentation of zinc in various brain regions,

especially in the hippocampal formation (Ebadi. 1991 and Ebadi et al., 1995) in adaptation to stress (Hidalgo

et al., 1994) as antioxidant agent (Hidalgo, et al., 1988; Lazo et al.. 1995; Sato and Bremer, 1993;

Thornalley and Vasak. 1985) etc. Thus, it has been suggested that MT protects against various oxidative

stress conditions (Sato and Bremner, 1993).

On the other hand, l-methyl-4-phenylpyridinium (MPP+) is the major metabolite of I-methyl-4-phenyl-

1,2,3,6_tetrahydropyridine (MPTP) (Chiba et al., 1984; Salach et al., 1984) a drug which when is

administered to non-human primates and mice, produces depletion of dopamine and cellular degeneration of

the nigrostriatal pathway, resembling the neurochemical deficits observed in idiopathic Parkinson’s disease

(Gerlach et al., 1991). It is now accepted that MPP+ is responsible of the neurotoxic effects of MPTP (Singer

and Ramsay, 1990).

MPTP is biotransformed to 1-methyl-4-phenylpyridinium ion (MPP+) by type-B monoamine oxidase (Chiba

et al., 1984). MPP+ is then accumulated into dopaminergic neurons by the high affinity dopamine uptake

system (Chiba et al., 1985) where it exerts its toxic effects by a mechanism still unknown. However, its

toxicity has been related to MPP+ inhibition of site I mitochondrial respiration (Nicklas et al., 1985; Ramsay

et al., 1987) and/or induction of oxidative stress (Adams and Odunze 1991; Chiueh et al.. 1994). In previous

studies, we found enhanced lipid peroxidation afler MPP+ administration to mice (Rojas and Rios, 1993) a

process dependent on overproduction of free radicals; we also found depletion of trace metals following

MPTP administration (Rios et al., 1995). MT is a protein important in both oxidative stress and trace metals

MT I+11 content after MPP+ neurotoxicity 145

metabolism. Recently, we found that MT inducers protect against MPTP neurotoxicity (Rojas and RioS,

1997). Therefore, the purpose of the present study was to evaluate the effect of MPP+ administration on MT

I + II protein content in the brain.

Methods

Adult Swiss albino mice NIH bred in-house, were used in all experiments. Animals weighed 25 to 30 g and

were up to 13 weeks old. Animals were fed with Purina chow (Purina Mexico) and drank water freely. The

room was kept dark between 7:00 P.M. and 7:00 A.M., temperature was 25 “C, and relative humidity 40%.

MPP+ iodine was obtained from Research Biochemicals Incorporated (RBI, Wayland, MA, U.S.A.) and

was dissolved in physiological saline solution (0.9% NaCI). All other reagents were from Merck, Mexico.

Exoerimental Procedure

MPP+, at a dose of 4.5, 9.0 or 18.0 a in 3 3 solution, was injected into the right lateral ventricle of mice,

as described previously by Haley and McCormick (1957). The la:t dose (18 j,g in 3 j,i solution) has been

shown to produce significant damage to dopaminergic neurons (Mihatsch et al., 1988). Animals similarly

injected with saline solution, served as controls, Mice from both groups were killed by cervical dislocation at

variable times (2 hr. 24 hr and 7 days) after MPP+ or saline were administered. Brains were immediately

removed and frozen at -75 “C. Striatum. midbrain, cerebellum, hippocampus and frontal cortex were

dissected out as described by Glowinski and lversen (1966) and weighed, and frozen again at -75 “C. For

dose-dependence assay, only corpus striatum and midbrain were studied. Later, the samples were

homogenized by sonication with 1 ml of 10 mM Tris-HCI, pH 8.2, containing 0.25 M sucrose, 2 rnM1

2-mercaptoethanol and 10 mM sodium azide. The homogenates were centrifuged at 50,OOOg for 1 hour at

4°C. The supernatants were stored at -75 “C. Tissue MT I+11 was analyzed by a highly specific and sensitive

radioimmunoassay as described previously (Gasull et al., 1993, 1994a).

146 P. Rojas et al.

Statistical Analvsis

Results were analyzed by the students t test or one-way analysis of variance, followed by Tukey’s test.

Values of p<O.O5 were considered of statistical significance.

No changes were observed in MT I+11 protein concentration in striatum. midbrarn, hippocampus, frontal

cortex and cerebellum 2 hrs as well as 7 days after MPP+ administration (data not shown). However, at 24

hrs after MPP+ intracerebroventricular administration into mice right lateral ventricles, MT I+11 protein

concentration increased by 33% in the midbrain and decreased by 34% in the striatum when compared with

the age-matched control mice (Fig.1). There was also an increase in MT I+11 protein concentration in the

cerebellum at 24 hrs after MPP+, probably related to stress induction (Gasull et al., 1994b ; Hidalgo et al.,

1990).

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Midbrain Striatum Hippocampus Cortex Cerebellum

Fig 1. MPP+, 18 pg/3 pl, or isotonic saline solution was injected into the lateral ventricles of mice, and their brains were removed 24 hrs later. The different brain regions were dissected out and later homogenized. MT-I and -II protein content in the different brain regions, was determined using an MT-I and -II protein- specific antibody in a highly sensitive radioimmunoassay. Results are expressed as mean +/- one standard error of mean, n = 9-i I. ‘Statistically significant difference from control, ~~0.05, Student’s t test. MPP+ = l- methyl-4-phenylpyridinium ion.

MT I+11 content after MPP’ neurotoxicity 147

MT I+11 protein level in the striatum was analyzed 24 hrs after intracerebroventricular injection of growing

doses of MPP+. There was a reduction in MT I+11 protein concentration, and it appeared to be dose-

dependent (Pig. 2). A similar analysis of MT I+11 protein level in the midbrain, following administration of the

same doses of MPP+, showed an increase in MT l+ll protein concentration (Fig. 3) statistically significant

only at the highest dose of MPP+ administered, 18.0 ~13 fl solution.

Striatum 1

Control MPP+ (4.5 pg) MPP+ (9 pg) MPP+ (18 pg)

Fig 2. MPP+ at 4.5, 9.0 or 18.0 pg/3 PI , or normal saline was injected into the lateral ventricles of mice, and their brains were removed 24 hrs later. The striatum was dissected out and later homogenized. MT-I and -II protein content in striatum was determined using an MT-I and -II protein-specific antibody in a highly sensitive radioimmunoassay. Results are expressed as mean +I- one standard error of mean, n = 9-11. * Statistically significant difference from control, p<O.O5. one way ANOVA followed by Tukey’s test. MPP+ * I- methyl+phenylpyridinium ion.

148 P. Rojas et al.

Discussion

The present study shows that MPP+, which generates free radicals, reduced MT I+11 concentration in

striatum. In a previous study (Rojas and Rios, 1997) we showed that MPTP administration also reduced the

concentration of total MT protein in striatum, suggesting that dopaminergic neurons that project from

substantia nigra into corpus striatum may influence the concentration of MT in this region. This also suggests

that MT-I and -II proteins may be associated with MPTP neurotoxicity, and this could be linked, with the pro-

oxidant effect of MPTP/MPP+ neurotoxicity (Rojas and Rios, 1993; Rojas and Rios. 1995; Rios and Tapia,

1987). Several exogenous agents induce MT expression in the brain, this suggests that the protection

observed in our previous study with the MT inducers, cadmium and dexamethasone, may be due to induced

MT-I and -II proteins.

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Midbrain

iVlPP+ (4.5 pg) MPP+ (9 pg) MPP+ (18 pg)

Fig 3. MPP+ at 4.5, 9.0 or 18.0 pg/3 pl, or normal saline was injected into the lateral ventricles of mice, and their brains were removed 24 hrs later. The midbrain was dissected out and later homogenized. MT-I and -II protein content in midbrain was determined using an MT-I and -II protein-specific antibody in a highly sensitive radioimmunoassay. Results are expressed as mean +I- one standard error of mean, n = 9-l I. l

Statistically significant difference from control, ~~0.05, one way ANOVA followed by Tukey’s test. MPP+ = l- methyl-%phenylpyridinium ion.

MT I+11 content after MPP+ neurotoxicity 149

On the other hand, it is well-known that induction of brain MT-I and MT-II by stress requires a period of 24

h (Hidalgo et al., 1988; Hidalgo et al., 1990; Hidalgo et al.. 1994). Thus this time was chosen to observe any

effect attributable to this factor. The measurement at 2 h was chosen on the basis of our previous finding of

increased in lipid peroxidation at this time (Rojas and Rios, 1993.).

Possible Explanations for the Findinas in Striatum

Several antioxidant defense systems prevent damage to tissues by oxygen radicals. These systems

include a range of specific antioxidants such as catalase for hydrogen peroxide, superoxide dismutase for

superoxide, glutathione peroxidase for hydrogen peroxide, and MT for hydroxyl radicals (Sato and Bremner,

1993). Indeed, transgenic mice deficient in MT-I and -II genes exhibit enhanced sensitivity to oxidative stress

(Lazo et al., 1995) suggesting that basal MT levels can function to regulate intracellular redox status in

mammalian cells.

The non-uniform induction of MT by MPP+ is not surprising in view of the fact that among four MT isoforms,

MT-III and MT-IV are less responsive to inducing agents and have a more restricted pattern of expressidn

(Palmiter et al., 1992; Kobayashi et al., 1993; Quaife et al., 1994). The lack of induction of striatal MT by

MPP+, which destroys dopaminergic terminals in striatum by forming free radicals, is remarkable for the

understanding of the pathophysiology of movement disorders. For example, indirect circumstantial evidence

supporting the formation of excess free radicals in the striatum of patients with Parkinson’s disease include

increased lipid peroxidation (Dexter et al., 1989; Warren et al., 1987). Since MT-I and -II proteins have

antioxidant properties (Sato and Bremner, 1993) their lack of induction in the striatum by MPP+, renders the

basal ganglia susceptible to high degrees of oxidative damage.

MT I+11 Concentration in Other Brain Reaions After MPP+ Administration

In the cerebellum MPTP does not appear to increase lipid peroxidation , increase hydroxyl radical

generation (Smith and Bennett, 1997) or decrease ATP concentration (Chan et al., 1991). However, MPTP

increases the expression of the immediate early gene c-fos (Duchemin et al., 1992) suggesting the

presence of cellular response to stress. MT proteins in the brain are induced by stress (Hidalgo et al., 1994)

this may explain the increase in MT I+11 proteins in the cerebellum in response to MPP+ administration.

There is no known clinical effect of MPP+ toxicity in the cerebellum.

1.50 P. Rojas et al.

In the midbrain, MPP+ increases lipid peroxidation (Rojas and Rios, 1993) and induces a transient

increase in c-fos expression (Duchemin et al., 1992). MPP+ causes progressive degeneration and loss of

dopaminergic neurons in the midbrain, an effect responsible for the parkinsonian syndrome caused by MPP+

administration. The induction of MT -I and II protein in the midbrain by MPP+ may be a protective mechanism

against the oxidative stress and neuronal loss caused by MPP+ administration.

In the present study , MPP+ had no effect on MT I+11 protein concentration in the hippocampus and frontal

cortex, suggesting that MT has no role, protective or otherwise, in either the toxicity MPP+ to these regions

of the brain.

General Considerations

It is possible that MT, which binds metals such as zinc (Ebadi et al., 1995) and copper, plays an important

role in protecting dopaminergic neurons from free radicals induced by MPTP. However, oxygen-derived free

radical species can directly interact with MT to oxidatively modify the protein (Jimenez et al., 1997) and the

loss of its antioxidant properties may lead to further oxidative stress. Oxidative stress is the result not only of

overproduction of reactive oxygen species, but also, it is a consequence of decreases in the cell’s

antioxidant protective mechanisms (Halliwell and Gutteridge. 1985). MPTP administration decreases the

level of reduced glutathione (Ferraro et al., 1986) and striatal MT content (Rojas and Rios, 1997), thereby

decreasing the cell’s thiol-related defenses against free radicals,

As a result of the high concentration of iron (prooxidant) and low concentration of ferritin (iron binding

protein) in the striatum, it is assumed that this region is highly vulnerable to oxidative stress, and this

vulnerability is increased by the reduction in MT concentration following MPP+ administration. MT may act,

therefore, as a sacrificial scavenger for hydroxyl and superoxide radicals. In addition, MPTP induces a

threefold increase in superoxide dismutase activity in the striatum (Thiffault et al., 1995) this may reflect a

compensatory change after MT reduction.

As stated previously it has been proposed that MT may participate in scavenging free radicals. Although

glutathione and glutathione peroxidase are important antioxidants in the body, the relative importance of their

contribution to antioxidant defense mechanisms in the brain is uncertain. For example, the rat brain has a

relatively low content of enzymes directed to protect against oxidative damage, such as glutathione

peroxidase and catalase and these enzymes are found predominantly in glia rather than neurons.

MT I+11 content after MPP+ neurotoxicity 151

It is quite possible, therefore, that MT plays a more important role in protecting dopaminergic neurons from

free radicals damage than is currently recognized.

This study shows that MPP+ decreases the concentration of MT-I and -II proteins in striatum. This

decreases may be associated with MPP+ neurotoxicity since this neurotoxin produces free radicals and MT

has been proposed as free radical scavenger.

Acknowledaements

This study was supported in part by CONACyT 0044P-M9506 and JH: CICYT SAF96-0189 grants. These

authors gratefully acknowledge the comments of Dr. Sultan Habeebu.

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Inquires and reprint requests should be addressed to:

Dr. Patricia Rojas

Laboratory of Neurotoxicology

lnstituto National de Neurologia y Neurocirugia,

Av. lnsurgentes Sur No. 3877 Col. La Fama, C.P. 14269

Mexico D.F., Mexico

Fax (52 5) 528 0095

e-mail: rojas@ buzon.main.conacyt.mx