Diethyldithiocarbamate and disulfiram inhibit MPP+ and dopamine uptake by striatal synaptosomes

European Journal of Pharmacology, 166 (1989) 23-29

23

Elsevier EJP 50858

Diethyldithiocarbamate and disulfiram inhibit MPP + and dopamine uptake by striatal synaptosomes Donato Di Monte *, Ian Irwin, Andreas Kupsch, Stephanie Cooper, Louis E. DeLanney and J. William Langston The Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128, and California Parkinson's Foundation, 2444 Moorpark Avenue, Suite 316, San Jose, CA 95128, U.S.A.

Received 8 December 1988, revised MS received 28 February 1989, accepted 11 April 1989

Diethyldithiocarbamate (DDC) was found to inhibit the uptake of both dopamine and 1-methyl-4-phenylpyridinium ion (MPP +, the putative toxic metabolite of the neurotoxicant MPTP) by striatal synaptosomes. Disulfiram, the corresponding disulfide of DDC, was effective at concentrations 1 000 times lower (10 -6 vs. 10 -3 M). Disulfiram, but not DDC, reacted very efficiently with synaptosomal protein thiols; both DDC- and disulfiram-induced uptake inhibition could be reversed by the thiol-reducing agent dithiothreitol. Two other thiol-reactive compounds, N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (PMB), also impaired the uptake of MPP ÷ by striatal synaptosomes. PMB, which does not cross membranes, was even more potent than the lipophilic NEM in blocking MPP ÷ uptake. These results suggest that (1) the effect of DDC may be mediated by disulfiram, (2) the uptake of MPP + and dopamine by striatal synaptosomes is dependent on the redox state of protein thiols, and (3) these protein thiols are located at the outer surface of synaptosomal membranes. Catecholamine uptake; MPP ÷ (1-methyl-4-phenylpyridinium ion); Dopamine; Diethyldithiocarbamate; Disulfiram

1. Introduction T h e neurotoxicity of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) is though to be dependent on the following sequence of events. First, the compound gains access into the central nervous system (CNS) by virtue of its lipophihc chemical structure (Langston et al., 1984; Markey et al., 1984). Once in the brain, M P T P is converted to its putative toxic metabolite, the 1methyl-4-phenylpyridinium species (MPP ÷) (Langston et al., 1984; Markey et al., 1984), by monoamine oxidase type B (MAO B) (Chiba et al.,

* To whom all correspondence should be addressed: The Institute for Medical Research, 2260 Clove Drive, San Jose, CA 95128, U.S.A.

1984). MPP ÷ is then thought to cause neuronal death after being accumulated within nigrostriatal neurons via the dopamine (DA) uptake system (Javitch et al., 1985). The discovery that diethyldithiocarbamate (DDC) exacerbates the toxic effects of M P T P in mice (Corsini et al., 1985) has provided a new tool for investigating the mechanism of action of this neurotoxin. It was first suggested (Corsini et al., 1985) that D D C may act by inhibiting superoxide dismutase (SOD), an enzyme essential to protect cells against oxygen radical-mediated damage (Heikkila et al., 1976). However, the role of oxygen radical formation in the cytotoxic effects of M P T P is still unclear (Di Monte and Smith, 1988), and D D C appears to affect MPTP-induced D A depletion at doses significantly lower (Corsini et al., 1985) than those necessary to inhibit SOD activity

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

24 in vivo (Heikkila et al., 1976). These observations suggest that other mechanisms are more likely to be involved in the enhancement of MPTP toxicity by D D C in mice. Indeed, D D C was recently found to (1) increase the delivery of MPTP to the CNS, (2) enhance the biotransformation of MPTP to MPP ÷, and (3) possibly reduce the clearance of MPP ÷ from the brain (Irwin et al., 1987). In light of the proposed sequence of events involved in MPTP neurotoxicity, it is also possible that D D C potentiates MPTP-induced DA depletion by enhancing uptake of MPP ÷ into dopaminergic terminals. ,In the present study, we tested this hypothesis; in addition, we extended our investigations to disulfiram (tetraethylthiuram disulfide), the corresponding disulfide of DDC, because the two compounds share some biological properties (Deitrich and Erwin, 1971; Forman et al., 1980) and are metabolically interconvertible (Strrmme, 1965).

2. Materials and methods

2.1. Chemicals N-[Methyl-3H]-4-phenylpyridinium acetate (88.4 Ci/mmol) and [3H]dopamine (27 C i / m m o l ) were purchased from New England Nuclear. DDC, disulfiram, dithiothreitol (DTT), N-ethylmaleimide (NEM), p-hydroxymercuribenzoate sodium salt (PMB) and 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) were all obtained from Sigma (St. Louis, MO). All solutions were prepared immediately before use. Disulfiram and N E M were dissolved in dimethylsulfoxide (DMSO) and added to the incubations at a final v / v of 0.2%. All other chemicals were reagent grade. 2.2. Synaptosome preparation Male C57B1/6 mice (age 7-10 weeks) were killed by cervical dislocation, their brains rapidly removed and dissected on ice. Striata from 5-10 animals were collected and immediately homogenized in 0.32 M sucrose (5% wt/v). The homogenate was centrifuged at 1000 x g for 10 rain and the resulting supernatant centrifuged at

27 000 × g for 30 min. The pellet obtained after this second centrifugation was suspended at 5 m g / m l (based on original wet weight) in buffer containing (mM) 50 Tris HC1, 120 NaC1, 5 KC1 and 10 glucose (pH 7.4). 2.3. Incubations All experiments were performed within 3 h of tissue preparation. A 1 ml aliquot of the striatal synaptosome preparation was added to 5 ml of buffer and incubated at 37 ° C for 10 min in the absence or presence of D D C or disulfiram. [3H] MPP + or [3H]DA were added (both at 5 nM final concentration) and striatal synaptosomes were incubated for an additional 10 min period. In experiments testing the effects of DTT, the initial preincubation period was extended to 20 min at which time DTT, with either [3H]MPP + or [3H] DA, was added. Incubations were stopped by placing the test tubes in ice. Samples were centrifuged at 27 000 × g for 30 min; supernatants were discarded and pellets washed twice with ice-cold buffer. Lastly, 2 ml absolute ethanol was added and, after 15 rnin, radioactivity was counted by liquid scintillation spectrometry in 1 ml of the extracts. In experiments where the effects of disulfiram and DDC on protein sulfhydryl groups were tested, synaptosomes were incubated at 1 mg p r o t e i n / m l for 20 min in the absence or presence of D D C or disulfiram. Concentrated perchloric acid (PCA) was added (5% v / v ) to stop reactions and precipitate proteins. 2.4. Biochemical assays Protein sulfhydryl groups were measured by the method of Di Monte et al. (1984) modified as follows. PCA was added to aliquots (2 ml) or incubations of striatal synaptosomes. After centrifugation, protein pellets were washed twice with 5% PCA and resuspended in 0.5 M Tris buffer (pH 8.0) containing 3% sodium dodecyl sulfate. D T N B (200 /~M final concentration) was then added and, after 15 min, the absorbance was measured at 412-520 nm. Data were expressed as nmol of sulfhydryl/mg protein, calculated on the basis of a calibration curve with reduced gluta-

25

thione (GSH). Protein concentration was determined by the method of Lowry et al. (1951).

c-

~'] control

o 450--

3. L Effects of DDC and disulfimm on M P P + and DA uptake by striatal synaptosomes Both D D C and disulfiram caused a concentration-dependent decrease in MPP + uptake by

~] dlsulfiram (2 pM) DDC (2 mM)

0~

3. Results

7

300-

E .2t¢

150K --i O

A .E E o150-

Fig. 2. Inhibition of dopamine uptake by disulfiram and DDC. Striatal synaptosomes were preincubated for 10 min with no addition (open bar) or in the presence of either 2 ttM disulfiram (striped bar) or 2 m M DDC (shaded bar). [3H]Dopamine (5 nM) was then added and incubations were carried on for an additional 10 min period. Non-specific uptake was measured in the presence of 10 /~M mazindol and subtracted. Values represent the means ( + S.D.) of three separate experiments.

~ioo-

-'~

5O-

÷ Q.

~

0

0

I

I

1.0 0.5 Disulfiram concentration,

I

2.0 ~.M

B

°t

o150

100

~.50

striatal synaptosomes (fig. 1A and B). Disulfiram was markedly more effective than its corresponding thiol, however, inhibiting MPP ÷ uptake by about 50% at a concentration as low as 1 / t M (fig. 1A). A similar effect was observed only after incubations with D D C at concentrations approximately 1 000 times greater (fig. 1B). D D C and disulfiram also decreased DA uptake by striatal synaptosomes (fig. 2). Again, these effects were obtained using concentrations of disulfiram far lower than those of DDC. Incubations with DMSO (final v / v = 0.2%), the solvent used with disulfiram, ruled out any effect of DMSO on MPP + or DA uptake (results not shown).

n.

IE

0

0

I

0.5 1.0 DDC concentration, mM

I

2.0

Fig. 1. Concentration-dependent inhibition of synaptosomal uptake of MPP + by disulfiram (A) and DDC (B). Striatal synaptosomes were preincubated in the presence of different concentrations of either disulfiram (striped bars) or DDC (shaded bars) for 10 rain before addition of 5 nM [3H]MPP+. Non-specific uptake was measured in the presence of 10 # M mazindol and subtracted. Values represent the means ( + S.D.) of four separate experiments.

3.2. Effects of disulfiram and D D C on protein sulfhydryl groups of synaptosomes The inhibition of MPP ÷ and DA uptake by disulfiram might be due to its interaction with protein thiols involved in the DA transport process (Deitrich and Erwin, 1971; Neims et al., 1966). Therefore, we next assessed the general ability of disulfiram to oxidize protein sulfhydryl

26

¢10o-

2 E o

m

E

e- 5 0 -

:E

ffl

e

a. 0

0

10

25 Disulfiram

50

100

concentration,

~M

Fig. 3. Effect of disulfiram on synaptosomal protein thiols. Striatal synaptosomes (1 m g p r o t e i n / m l ) were incubated for 20 min in the presence of different concentrations of disulfiram. At this time, reactions were stopped by addition of 60% PCA and protein sulfhydryl groups were measured as described in the Materials and methods section. Values represent the m e a n s (4- S.D.) of three separate experiments. Statistical significance was determined by simple one-way analysis of variance (ANOVA) which showed an F value of 576.6 (P < 0.001).

groups of striatal synaptosomes. Incubations of synaptosomes (1 mg p r o t e i n / m l for 20 min) in the presence of different concentrations of disulfiram resulted in a dose-dependent loss of protein thiols (fig. 3). The ratio of nmol of disulfiram a d d e d / nmol of protein-SH groups lost was 1 : 1 at concentrations up to 50 #M.

Addition of D D C to synaptosome incubations also induced a significant decrease in protein sulfhydryl groups (table 1). This effect, however,

['1 ¢:

i

TABLE 1 Effects of D T T on the loss of protein thiols induced by D D C and disulfiram. Striatal synaptosomes were preincubated (1 mg p r o t e i n / m l ) for 20 rain in the absence or in the presence of either D D C (5 raM) or disulfiram (50 #M). At this time, 1 m M D T T was added to some samples and incubations were carried on for an additional 10 rain period. Reactions were stopped by addition of 60~ PCA and protein sulfhydryl groups were measured as described in the Materials and methods section. Values represent the m e a n s (4- S.D.) of three separate experiments and are expressed as nmoi of s u l f h y d r y l / m g protein, calculated on the basis of a calibration curve with GSH. Addition

Protein sulfhydryl groups

None D T T (1 mM) D D C (5 mM) DDC + DTT Disulfiram (50/~M) Disulfiram + D T T

86.0 + 2.3 88.2 + 1.8 60.4+2.7 a 87.5 + 2.9 37:5 + 0.9 a 81.9 4- 3.7

" Significantly different from control (P < 0.001).

]1

E

without

DT'r

II .......

l

o

t~

n4n

Control

Disulfiram (2 p.M)

DDC (2 mM)

Fig. 4. Effects of D T T on the inhibition of M P P + uptake by disulfiram and DDC. Striatal synaptosomes were incubated for 20 min with no addition or in the presence of either disulfiram (2 /~M) or D D C (2 raM). At this time, [3H]MPP + (5 n M ) was added with (open bars) or without (solid bars) 0.5 m M D T T and incubations were carried on for an additional 10 min period. Non-specific uptake was measured in the presence of 10 F M mazindol and subtracted. Values represent the m e a n s ( + S.D.) of three separate experiments.

27

was obtained using concentrations of D D C (5 mM) much higher than those of disulfiram.

3.3. Effects of DTT on uptake inhibition by disulfiram and DDC If the inhibition of catecholamine uptake by D D C and disulfiram is due to formation of mixed disulfides, then DTT, a strong thiol-reducing agent (Bellomo et al., 1987), might be expected to reverse this effect. This proved to be the case, as (1) the levels of protein thiols of synaptosomes preincubated for 20 rain in the presence of either disulfiram or D D C resulted in values similar to control when measured 10 min after addition of D T T (table 1), and (2) D T T treatment reversed the inhibition of MPP ÷ uptake by both disulfiram and D D C in striatal synaptosomes (fig. 4). It is important to note that the action of D T T cannot be attributed to a direct interaction with disu l f i r a m / D D C , as the thiol-reducing agent was added after allowing disulfiram and D D C to exert their toxic effects. D T T was similarly effective in reversing the inhibition of DA uptake by these two compounds (data not shown).

3.4. Effects of other thiol-reactive agents on MPP ÷ uptake by striatal synaptosomes N E M is a lipophilic, alkylating agent that can cross membranes and form addition products with sulfhydryl compounds (Jacob and Jandl, 1962). PMB also reacts with thiols by forming mercapTABLE 2 Effects of N E M and PMB on the uptake of M P P + by striatal synaptosomes. Synaptosomes were preincubated for 10 min in the absence or in the presence of either N E M (20/zM) or PMB (10/~M). [3H]MPP+ (5 nM) was then added, and incubations were carried on for an additional 10 min period. Values represent the m e a n s (4- S.D.) of three separate experiments and are expressed as p m o l / g tissue per 10 re.in. Non-specific uptake was measured in the presence of 10 /~M mazindol and subtracted. Addition

M P P + uptake

None N E M (20/*M) PMB (10 # M )

118.74-15.2 29.54- 6.6 14.64- 6.1

tides but, as a charged molecule, does not easily permeate intact membranes (Jacob and Jandl, 1962). We used both of these compounds to further assess the dependency of MPP ÷ uptake on sulfhydryl groups, and to learn more about the localization of thiols sensitive to inhibition (i.e. are they within or outside synaptosome membranes?). Both N E M and PMB inhibited MPP ÷ uptake, the latter being effective at a lower concentration (table 2).

4. Discussion

Rather than supporting our initial hypothesis that D D C would enhance uptake of MPP + into striatal synaptosomes, these experiments showed that uptake of both MPP + and dopamine were actually inhibited by D D C at millimolar concentrations. MPP ÷ uptake via the dopaminergic system is thought to be one of the essential steps leading to MPTP-induced neurotoxicity (Javitch et al., 1985; Chiba et al., 1985; Sundstrom and Jonsson, 1985; Ricaurte et al., 1985; Mayer et al., 1986; Melamed et al., 1985). It is not clear, therefore, how D D C might enhance M P T P neurotoxicity in vivo while impairing the uptake-mediated entrance of MPP ÷ into dopaminergic terminals. It will be important to determine if these in vitro observations can be confirmed in vivo. The concentrations of D D C used to block synaptosomal uptake would be reached in vivo if the dose of D D C that enhances MPTP toxicity (400 m g / k g ; Irwin et al., 1987) is evenly distributed throughout the total body water (Forman et al., 1980). On the other hand, it is conceivable that the distribution of D D C within the brain might be uneven, making its concentration near the uptake site not high enough to affect dopaminergic uptake. A second intriguing finding in our in vitro system was that uptake inhibition by disulfiram was achieved at concentrations 1000 times lower than DDC, raising the possibility that disulfiram may play a role in mediating the effect of DDC. Contamination of D D C with disulfiram has already been proposed to explain the slight effects induced by D D C on enzymatic activities which

28 are strongly i n h i b i t e d b y d i s u l f i r a m ( D e i t r i c h a n d Erwin, 1971). T h e fact t h a t d i s u l f i r a m is a b l e to b l o c k M P P + a n d D A u p t a k e b y striatal s y n a p t o somes at a c o n c e n t r a t i o n as low as 10 - 6 M m a k e s o u r in vitro results m o r e relevant in terms of their p o s s i b l e o c c u r r e n c e in vivo. Evidence exists for the in vivo c o n v e r s i o n of D D C to disulfiram, since a d m i n i s t r a t i o n of D D C results in the f o r m a t i o n of m i x e d disulfides with p l a s m a a n d tissue p r o t e i n s (Str/Smme, 1965). This r e a c t i o n is likely to require the disulfide b o n d of d i s u l f i r a m (StriSmme, 1965) and, as discussed below, is also likely to b e involved in the i n h i b i t i o n of M P P ÷ a n d D A u p t a k e b y D D C a n d disulfiram. W h e t h e r o r n o t disulfiram is a c t u a l l y the m e d i a t o r o f one or m o r e of the effects o f D D C on M P T P toxicity requires further study, b u t the question is i m p o r t a n t to pursue, as d i s u l f i r a m is c u r r e n t l y used in clinical practice, a n d has b e e n r e p o r t e d to cause a v a r i e t y o f neurological a b n o r m a l i t i e s in h u m a n s ( H o t s o n a n d L a n g s t o n , 1976). O u r last set of e x p e r i m e n t s was d e s i g n e d to e x p l o r e the m e c h a n i s m o f a c t i o n b y w h i c h syna p t o s o m a l u p t a k e is inhibited. W e f o u n d that low c o n c e n t r a t i o n s of d i s u l f i r a m r e a c t e d r a p i d l y a n d stoichiometrically with synaptosomal protein thiols. Similar effects were seen w i t h D D C , b u t at m u c h higher c o n c e n t r a t i o n s . F u r t h e r , the thiol-red u c i n g agent D T T effectively reversed the inhibition of M P P ÷ a n d D A u p t a k e i n d u c e d b y b o t h d i s u l f i r a m a n d D D C . T h e s e results are c o n s i s t e n t with earlier studies i n d i c a t i n g that m o s t of the effects of d i s u l f i r a m on enzymes a n d cellular processes are the result of its reactivity t o w a r d p r o t e i n thiols ( N e i m s et al., 1966; Hassinen, 1966). T h e y p r o v i d e the first evidence that M P P + accum u l a t i o n is d e p e n d e n t o n the r e d o x state of p r o tein thiols (i.e. b l o c k a d e of these thiols, either b y o x i d a t i o n or alkylation, results in u p t a k e inhibition). This o b s e r v a t i o n c o u l d l e a d to a new experim e n t a l p a r a d i g m for the s t u d y of the u p t a k e system, since p r o t e i n thiols can b e altered b y a variety of agents a n d c o n d i t i o n s , such as the g e n e r a t i o n of o x y g e n radicals ( D i M o n t e et al., 1984). T h e a b i l i t y of d i s u l f i r a m to b l o c k M P P ÷ a n d D A u p t a k e at a c o n c e n t r a t i o n as low as 2 g M indicates that thiol g r o u p s critical for this u p t a k e are relatively ' e x p o s e d ' to reactive c o m p o u n d s .

Disulfiram may inhibit uptake by forming mixed disulfides at the o u t e r surface of s y n a p t o s o m a l m e m b r a n e s . This h y p o t h e s i s is s u p p o r t e d b y d a t a o b t a i n e d using o t h e r thiol-reactive c o m p o u n d s ; the h y d r o p h i l i c m o l e c u l e o f P M B , which w o u l d n o t b e e x p e c t e d to cross m e m b r a n e s at the conc e n t r a t i o n s used ( J a c o b a n d J a n d l , 1962), was f o u n d to b e m o r e active t h a n the l i p o p h i l i c N E M in b l o c k i n g M P P ÷ u p t a k e .

Acknowledgements The authors wish to thank John Skratt and David Remmler for technical assistance, and to gratefully acknowledge David Rosner and Pamela Schmidt for their help in the preparation of this manuscript. Dr. Di Monte is a recipient of the Lillian Schorr Research Fellowship from the Parkinson's Disease Foundation, New York, NY. This work was supported in part by the California Parkinson's Foundation, the United Parkinson Foundation, the Parkinson's Disease Foundation, and the National Institute of Aging (R01 AG07348-01).

References Bellomo, G., F. Mirabelli, D. Di Monte, P. Richelmi, H. Thor, C. Orrenius and S. Orrenius, 1987, Formation and reduction of glutathione-protein mixed disulfides during oxidative stress, Biochem. Pharmacol. 36, 1313. Chiba, K., A.J. Trevor and N. Castagnoli, Jr., 1984, Metabolism of the neurotoxic tertiary amine, MPTP, by brain monoamine oxidase, Biochem. Biophys. Res. Commun. 120, 574. Chiba, K., A.J. Trevor and N. Castagnoli, Jr., 1985, Active uptake of MPP +, a metabofite of MPTP, by brain synaptosomes, Biochem. Biophys. Res. Commun. 128, 1229. Corsini, G.U., S. Pintus, C.C. Chiueh, J.F. Weiss and I.J. Kopin, 1985, 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice is enhanced by pretreatment with diethyldithiocarbamate, European J. Pharmacol. 119, 127. Deitrich, R.A. and V.G. Erwin, 1971, Mechanism of the inhibition of aldehyde dehydrogenase in vivo by disulfiram and diethyldithiocarbamate, Mol. Pharmacol. 7, 301. Di Monte, D., D. Ross, G. Bellomo, L. Ekl~Swand S. Orrenius, 1984, Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes, Arch. Biochem. Biophys. 235, 334. Di Monte, D. and M.T. Smith, 1988, Free radicals, hpid peroxidation and 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism, in: Reviews in the Neurosciences, eds. C.D. Marsden, P. Jenner and E.G. Jones (Freund Publishing House, Ltd., London) p. 67.

29 Forman, H.J., J.L. York and A.B. Fisher, 1980, Mechanism for the potentiation of oxygen toxicity by disulfiram, J. Pharmacol. Exp. Ther. 212, 452. Hassinen, I., 1966, Effect of disulfiram (tetraethylthiuram disulfide) on mitochondrial oxidations, Biochem. Pharmacol. 15, 1147. Heikkila, R.E., F.S. Cabbat and G. Cohen, 1976, In vivo inhibition of superoxide dismutase in mice by diethyldithiocarbamate, J. Biol. Chem. 251, 2182. Hotson, J.R. and J.W. Langston, 1976, Disulfiram-induced encephalopathy, Arch. Neurol. 33, 141. Irwin, I., E.Y. Wu, L.E. DeLanney, A. Trevor and J.W. Langston, 1987, The effect of diethyldithiocarbamate (DDC) on the biodisposition of MPTP: An explanation for enhanced neurotoxicity, European J. Pharmacol. 141, 209. Jacob, H.S. and J.H. Jandl, 1962, Effects of sulfhydryl inhibition on red blood cells. I. Mechanism of hemolysis, J. Clin. Invest. 41, 779. Javitch, J.A., R.J. D'Amato, S.M. Strittmatter and S.H. Snyder, 1985, Parkinsonism-inducing neurotoxin, 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine: Uptake of the metabolite N-methyl-4-phenylpyridine by dopamine neurons explains selective toxicity, Proc. Natl. Acad. Sci. U.S.A. 82, 2173. Langston, J.W., I. Irwin, E.B. Langston and L.S. Fomo, 1984, 1-Methyl-4-phenyl-pyridinium ion (MPP + ): Identification of a metabolite of MPTP, a toxin selective to the substantia nigra, Neurosci. Lett. 48, 87. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265.

Markey, S.P., J.N. Joharmessen, C.C. Chiueh, R.S. Bums and M.A. Herkenham, 1984, Intranenronal generation of a pyridinium metabolite may cause drug-induced parkinsonism, Nature 311, 464. Mayer, R.A., M.V. Kindt and R.E. Heikkila, 1986, Prevention of the nigrostriatal toxicity of 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine by inhibitors of 3,4-dihydroxyphenylethylamine transport, J. Neurochem. 47, 1073. Melamed, E., J. Rosenthal, O. Cohen, M. Globus and A. Uzzan, 1985, Dopamine but not norepinephrine or serotonin uptake inhibitors protect mice against neurotoxicity of MPTP, European J. Pharmacol. 116, 179. Neims, A.H., D.S. Coffey and L. Hellerman, 1966, Interaction between tetraethylthiuram disulfide and the sulfhydryl groups of d-amino acid oxidase and of hemoglobin, J. Biol. Chem. 241, 5941. Ricaurte, G.A., J.W. Langston, L.E. DeLarmey, I. Irwin and J.D. Brooks, 1985, Dopamine uptake blockers protect against the dopamine depleting effect of 1-methyl-4phenyl-l,2,3,6-tetrahydropyridine (MPTP) in the mouse striatum, Neurosci. Lett. 59, 259. StriSmme, J.H., 1965, Metabolism of disulfiram and diethyldithiocarbamate in rats with demonstration of an in vivo ethanol-induced inhibition of the glucuronic acid conjugation of the thiol, Biochem. Pharmacol. 14, 393. Sundstrom, E. and G. Jonsson, 1985, Pharmacological interference with the neurotoxic action of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) on central catecholamine neurons in the mouse, European J. Pharmacol. 110, 293.