Role of intracellular Cd2+ in catecholamine release and lethality in PC12 cells

Toxicology Letters ELSEVIER

Toxicology Letters 81 (1995) 151-157

Role of intracellular Cd*+ in catecholamine release and lethality in PC12 cells Anumantha

G. Kanthasamya’b,

Gary E. Isoma, Joseph

L. Borowitz*”

‘Departmen of Pharmacology and Toxicology, Purdue University. West Lafayette, IN 47907-1334. USA bParkinson and Movemenf Disorder Laboratory, Department of Neurology, University of California, Irvine. Irvine. CA 92717, USA

Received I5 February 1995;revision received I2 June 1995;accepted I3 June 1995

To evaluate the role of intracellular Cd2+ in catecholamine release and lethality in rat pheochromocytoma (PC12) cells the following results were obtained: (11 the presence of Cd2’ intracellularly was demonstrated with the Cd2+sensitive fluorescent dye BTC-SN, [2] Cd’+ entry through Ca’+channels was either blocked with nifedipine or diltiazem or increased with Bay K8644, [3] Cd2+ entry through voltage sensitive Ca2+ channels was related to dopamine release and cell lethality, [4] a calmodulin inhibitor protected against Cd2+ toxicity, and [5] extracellular Ca2+ concentration, altered prior to Cd’+ exposure, was inversely related to dopamine release by Cd’+. The data indicate intracellular effects of Cd2+ rather than cell surface actions are primarily involved in neurotransmitter release and lethality by toxic levels of Cd” in adrenomedullary cells.To evaluate the role of intracellular Cd’+ in catecholamine release and lethality in rat pheochromocytoma (PC12) cells the following results were obtained: [l] the presence of Cd’+ intracellularly was demonstrated with the Cd2+-sensitive fluorescent dye BTC-SN, [2] Cd” entry through Ca’+-channels was either blocked with nifedipine or diltiazem or increased with Bay K8644, [3] Cd’+ entry through voltage sensitive Ca 2+channels was related to dopamine release and cell lethality, [4] a calmodulin inhibitor protected against Cd2+ toxicity, and [S] extracellular Ca2+ concentration, altered prior to Cd2+ exposure, was inversely related to dopamine release by Cd’+. The data indicate intracellular effects of Cd’+ rather than cell surface actions are primarily involved in neurotransmitter release and lethality by toxic levels of Cd2+ in adrenomedullary cells. Keywords: Cadmium; Neurotransmitter

release; Calcium channels; Calmodulin; PC12 cells; Neurotoxicity

1. Introduction

Several mechanisms have been reported to explain the cellular actions of Cd2+: activation of an * Corresponding author, Tel.: +I 317 4941407; Fax: +I 317 4941414.

receptor followed by IP3 formation and mobilization of intracellular Ca2+ [l], an action on cell surfaces to allow Na+ entry, cause extracellular

depolarization and opening Ca2+ channels [2], Cd2+ entry nels to mobilize intracellular entry through Ca *+ channels

0378-4274/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-4274(95)03425-K

of voltage-sensitive through Ca*+chanCat+ [2], and Cd’+ followed by a direct

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action of Cd’+ on intracellular structures [3,4,5]. The relative contributions of these four cellular actions of Cd2+ to a given response have not been fully evaluated in any cell type. The present study is an attempt to estimate the importance of intracellular Cd’+ in catecholamine release and cell death in rat pheochromocytoma (PC12) cells, an extensively used neuronal model [6]. 2. Materials and methods 2.1. Cell culture

PC12 cells were obtained from ATCC (Rockville, MD) and propagated by serial passage. Cells were cultured as monolayers in RPM1 1640 medium with 10% heat inactivated horse serum, 5% fetal calf serum and 1% penicillin/streptomycin. Cells were maintained at 37°C in 95% air and 5% CO2 atmosphere. Since PC12 cells mutate on prolonged passage [6], cells were recultured from stock every 2-3 months or within 20 passages. Functional properties of the new cells, such as ability to release neurotransmitter and ion influxes on depolarization, were verified. Cells from 4 to 5 days old cultures were used in the experiments. 2.2. Treatment Cells (1.5-2.0 x lo6 ml) were suspended in calcium (1.2 mM) containing Krebs-Ringer (Ca2+KR) buffer or calcium-free Krebs-Ringer (Cadeficient KR) and incubated with various concentrations of CdC12 at 37°C for 40 min. Drugs used to block voltage-sensitive calcium channels or to block calmodulin-dependent processes were added to the incubation buffer 15 min before treatment with Cd*+. The voltage-sensitive calcium channel agonist Bay K 8644 was dissolved in ethanol and included in Ca*+-deficient KR buffer containing 35 mM KCl. After incubation, the supernatant was separated by centrifugation (9000 g x 10 min) and used for measurement of neurotransmitter release. A 50 ~1 aliquot of ice-cold antioxidant solution containing 0.1% sodium bisulfite and 0.5% EDTA in 0.1 M perchloric acid was added to 450 @Iof sample. The cell pellet was lysed with antioxidant solution to measure the intracellular neurotransmitter level. Samples were purged with

nitrogen and frozen until assayed for catecholamines. 2.3. Dopamine (DA) assay DA was analyzed by high performance liquid chromatography (HPLC) on a C-18 reversed phase column (3 nun x 100 mM) with the flow rate set at 1 ml/min isocratically. The HPLC system consisted of 1lOB Beckman (San Ramon, CA) pump coupled with a 427A integrator (Beckman, San Ramon, CA) and a refrigerated autosampler (Tosohaas, Philadelphia, PA). DA was estimated by electrochemical detection using a glassy-carbon working electrode and an Ag/AgCl reference electrode (Bioanalytical Systems, West Lafayette, IN) with the electrode oxidation potential maintained at +650 mV. The mobile phase consisted of monochloroacetic acid, 0.15 M; sodium octyl sulfonate, 0.13 mM; disodium EDTA 0.67 mM; sodium hydroxide, 0.12 M and 1.5% acetonitrile. The pH of the buffer was adjusted to 3.1 with phosphoric acid and the solution was filtered and degassed. Dihydroxybenzylamine (DHBA) was used as the internal standard. 2.4. Fluorescence assay of intracellular Cd2+ Cd2+ influx into PC12 cells was measured by monitoring fluorescence of the heavy metalsensitive fluorescent probe, BTC-5N. Cells were washed and resuspended in Ca*+-deficient KR buffer and they were loaded with I,uM BTC-5N /AM (final concentration) for 30 min at room temperature. After loading, the cells were washed twice and the cell concentration was adjusted to 2.0 x lo6 cells /ml. Aliquots were treated with test compounds for 40 min in brown tubes and incubated at 37°C in a thermomixer (Brinkmann Instruments, Inc., Westbury, NY). After incubation, cells were washed once and fluorescence measured at EX 415 and EM 500 nm (F-2000, Hitachi Instruments. Inc., San Jose, CA). The fluorescence from untreated cells was subtracted from the treatment groups to obtain changes in fluorescence. 2.5. Cell viability

Cell viability was determined by the dye exclusion method [7] with slight modifications. Cell

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suspensions in Ca’+-KR buffer or Ca2+-deficient KR were incubated with Cd? alone or Cd’+ with pre-treatment drugs at 37°C for 3 h. Aliquots (0.2 ml) were withdrawn at 60 and 180 min from the suspension and diluted with 0.2% trypan blue. Cells excluding dye and cells stained were counted in a hemocytometer using an inverted microscope. Cell viability was expressed as percent cell death. 2.6. Chemicals Bay K 8644, W-7 and nifedipine were purchased from Research Biochemicals International (Natick, MA). Dopamine hydrochloride, DHBA and sodium-octyl-sulfate were purchased from Bioanalytical Systems Inc., (West Lafayette, IN). The fluorescent probe BTC-5N was obtained from Molecular Probes, Inc. (Eugene, OR). Cadmium chloride and monochloroacetic acid were purchased from Mallinkroft Co., (St. Louis, MO) and all other chemicals were obtained from Sigma Chemical Co., (St. Louis, MO).

:

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200 Cd*+

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2.7. Statistics

Data were analysed by ANOVA with Student Newman-Keuls as a post hoc test to determine differences between groups. Differences less than P = 0.05 were accepteo as significant. 3. Results 3.1. Cd’+-induced dopamine (DA) presence and absence of Ca2+

release in the

To examine the effect of extracellular Ca2+ on the response of PC12 cells to graded concentrations of Cd’+, Ca2+ was either included in or omitted from the medium. DA release by Cd2” was actually greater when extracellular Ca2+ was omitted (Fig. 1). Thus involvement of extracellular Ca2+ alone cannot fully explain Cd2+-induced adrenomedullary secretion. 3.2. Time course of Cd2+-induced DA release

Onset of DA release by Cd’+ is rapid (within minutes) both in the presence and absence of extracellular Ca 2+ (Fig. 2). It appe ars that Ca2+dependent secretion occurs prominently on initial exposure whereas DA release in the absence of

600

(PM)

Fig. 1. Effect of Cd*+ on dopamine (DA) release from PC12 ceils. Cells were treated with CdCl, for 40 min at 37°C and DA released into the supematant was determined. Squares and circles indicate Cdz+-induced DA release in Ca*+-deficient and Ca*+containing medium, respectively. Asterisk indicates significant (*P
k!

0

40

10 MIN %ER

50

c3:*+

Fig. 2. Time-course of DA release induced by Cd” from PC 12 cells. Cells were treated with 100 PM CdClz and extracellular DA was measured. Squares and circles indicate Cd”-induced DA release in Ca’+-deficient and Ca’+-containing medium. respectively. Asterisk indicates significant (*P
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74 to 191% above control when Bay K 8644 was

present without calcium in the medium (Fig. 4). Obviously, movement of Cd2+ through Ca2+ channels in PC12 cells has a profound effect on cellular function. 5

20

> 0 I-

10

i

*

I

Ill

P

2

0

’ Cd*+

Cd*+ +Dtz

Cd *+ +Nif

Fig. 3. Effect of Ca2+ channel blockers, nifedipine (Nif) (I pM), or diltiazem (Dtz) (25 PM) on Cd’+-induced DA release. Cells were pretreated with the blockers for 15min and incubation continued for an additional 40 min after inclusion of 100 pM CdCI, after which time DA release was measured. Values are means f S.E.M. from four experiments done in Ca-KR buffer. Asterisks indicate a significant difference (*P< 0.05) from cells treated with Cd2+ alone.

[Ca2’], is more prominent on prolonged exposure (Fig. 2). These results suggest that at least two different mechanisms are involved in the response of PC12 cells to Cd2+.

3.5. Measurement of intracellular Cd’+ Direct measurement of Cd2+ in PC12 cells can be made using BTC-SN a Cd2+-sensitive fluorescent dye. When cells were loaded with BTC-5N and extracellular dye removed by washing, the increases in fluorescence after Cd2+ addition were proportional to Cd2+ in the cells. Fig. 5 shows that treatment of PC12 cells with Cd2+ alone caused a considerable increase in BTC-5N fluorescence in a Ca2+-deficient medium and the increase in fluorescence is reduced by about 50% using Ca2+ channel blockers. Assuming a proportionality and complete channel blockade, it appears that about half of the Cd2+ which enters PC12 cells in the absence of Ca2+ passes through Ca 2+ channels. Furthermore, treatment with Bay K 8644, the Ca2+ channel agonist, allows more Cd2+ to enter PC12 cells as reflected by the greatly enhanced BTC-5N fluorescence (Fig. 5). This is the first unequivocal demon-

3.3. Importance of intracellular Cd’+ in DA secretion from PC12 cells

About 65% of the Cd2+-induced catecholamine secretion was blocked by Ca2+ channel blockers (Fig. 3). Thus about two-thirds of the DA release under these conditions results from the presence of Cd2+ inside PC12 cells. A substantial part of the response of PC12 cells to Cd2+ involves voltagesensitive Ca2+ channels. Cd2+ apparently enters PC12 cells via calcium channels and initiates secretion primarily by virtue of its presence inside cells. 3.4. Effect of a Ca2+ channel agonist on Cd2+induced DA release

To further establish that Cd” does indeed pass through voltage-sensitive Ca2+ channels in PC 12 cells and to estimate roughly the extent of involvement of these channels in Cd2+-induced DA release, the Ca2+ channel agonist Bay K 8644 was employed. Cd2+-induced secretion increased from

!i :

2 a.

50

~

i

1~

I

0 Cd

2+

Bay K

Bay K and

Cd*+

Fig. 4. Effects of the calcium channel agonist Bay K 8644 on Cd’+-induced DA release from PC12 cells. Cells were pretreated with I pM Bay K 8644 for IS min and the incubation continued for an additional 40 min after inclusion of 100 pM CdCI, at which time DA release was measured. Values are means f S.E.M. from four experiments done in Ca2+-deficient buffer. Asterisks indicate a significant difference (*P
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Letters 81 (1995) 151-157

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and 5mM Ca2

*

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Fig. 5. Effect of nifedipine (1 PM), diltiaxem (25 ah4) and Bay K 8644(1 PM) on Cd*+ influx. PC12 cells were loaded with BTC-SN and preincubated with the test compounds for I5 mm and the incubation continued for an additional 40 mitt after addition of CdC12 (100 PM). Fluorescence was monitored at EX 415 and EM 500 nm. Values are. means f S.E.M. from four experiments done in Ca*+-deBcient buffer. Asterisks indicate a significant difference (*P
60

min

180

min

Fig. 7. Effects of 1 PM Bay K 8644 and 5 mM Ca*+ on Cd’+induced cell death. PC12 cells were preincubated with the test compounds for I5 min and incubation continued for an additional 180 min. after inclusion of CdC12 (100 PM). Cell viability was monitored at 60 and 180mm intervals using @pan-blue. Values are means + S.E.M. from four experiments done in Ca*+deficient buffer. Asterisks indicate a significant difference (*P
stration using a fluorescent dye insensitive to Ca2+ and Mg2+ that Cd2+accumulates inside PC12 cells.

a

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+and W7

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Fig. 6. Effect of nifedipine (1 pM), diltiaxem (25 PM), or W-7 (IO PM) on Cd*+-induced cell death. PC12 cells were preincubated with the test compounds for IS min and the incubation continued for an additional 60 to 180 mitt after addition of CdCI, (100 CM). Cell viability was monitored at 60 and 180 min intervals using trypan-blue. Values are means f S.E.M. from four experiments done in Ca *+ KR butTer. Asterisks indicate a significant difference (*P
3.6. Role of Ca2+ channels in Cd2’ toxicity That Cd’+ entry into PC12 cells through Ca2+ channels is a factor in Cd2+ toxicity, is demonstrated in Fig. 6. Voltage-sensitive Ca2+ channel blockers provide strong protection for PC12 cells against Cd2+ in a Ca2+containing medium. A possible intracellular site of action of Cd’+ is calmodulin since W-7, a calmodulin inhibitor, protected the cells against Cd2+ (Fig. 6). In a Ca2+-deficient medium, Cd2+ toxicity is enhanced by the Cachannel agonist Bay K 8644 (Fig. 7). Thus Cd2+ which penetrates through Ca2+ channels in PC12 cells is sufficient to mediate cell death and extracellular Ca2+ is unnecessary for this effect to occur. Actually, Cd2+ toxicity is attenuated by a high Ca2+ medium (Fig. 7) no doubt by competitive inhibition of Cd2+ entry into the cells. 4. Discussion Cd2+ blocks Ca2+ channels in neurons but it is

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relatively ineffective in blocking Ca2+ channels in adrenal medulla [8]. Therefore, it is advantageous to use adrenomedullary cells to study Cd2+-evoked catecholamine release. The ability of Cd2+ to release adrenal catecholamines has been known since 1974 [9]. Recent work has shown clearly that Cd2+ acts both inside the cell and on the cell surface to cause adrenomedullary secretion. Yamagami et al. [2] reported that the action of l-10 PM Cd2+ on the surface of cat adrenomedullary cells still occurs even if intracellular Cd2+ is chelated by TPEN. Yamagami et al. [2] further showed that Cd’+ acts on the cell surface to allow Na+ entry, depolarization and opening of Ca2’ channels followed by accumulation of Ca2+. Only after prolonged exposure (several min) to Cd2+ does sufficient accumulation of intracellular Cd2+ occur with associated catecholamine release [2]. Present results extend this work to demonstrate that the delayed effect associated with elevated intracellular Cd2+ is stronger than the extracellular effect in terms of the extent of evoked secretion. The mechanism by which intracellular Cd2+ causes adrenal catecholamine release is not clear. Yamagami et al. [2] suggest that Cd2+ mobilizes intracellular calcium and that this effect is blocked by Zn2+. On the other hand, there is evidence that Cd2+ might increase exocytosis independent of Ca’+. According 1y Cd2+ activates calmodulin [3] which phosphorylates synapsin to enhance catecholamine release [lo]. Furthermore the calmodulin inhibitor ‘W-7’ protects against Cd2+ poisoning in vivo [ 11) as well as in the in vitro system in the present study. Most likely Cd2+ mobilizes intracellular Ca2+ [12] and also has direct intracellular actions of its own to evoke adrenomedullary secretion. Cd2+ not only initiates catecholamine secretion from adrenomedullary tissue [ 121but also releases neurotransmitter from peripheral nerves [ 13,14,15]. To our knowledge, studies of Cd2+-induced brain neurotransmitter release have not been done but damage to brain synaptosomal membranes does occur in the presence of Cd’+ [16]. Cd2+ also causes deficits in olfactory function [ 17,181and neuronal injury [19]. Normally the blood-brain barrier is not very permeable to Cd2+, and the major toxic effects of chronic Cd2+ exposure are on the kidney. However

blood-brain barrier permeability is increased by ethanol and Cd2+ penetration into the brain is increased several fold in ethanol treated animals [20]. Blood-brain barrier permeability is also enhanced by bacterial collagenase [21] and Cd2+ would be expected to penetrate into the brain more readily in acute cerebral inflammation [22]. Furthermore high blood pressure [23] and certain drugs (haloperidol, chlorpromazine) [24] increase barrier permeability and may also allow greater Cd2+ entry. Intracellular sites of action of Cd2+ may be of great significance when Cd2+ penetration into the brain is enhanced. In conclusion, low concentrations of Cd2+ appear to act on the surface of neuronal type cells to activate secretory mechanisms [2]. Higher toxic concentrations allow Cd’+ to enter neuronal cells mainly by way of Ca 2+ channels. Cd2+ mobilizes intracellular Ca2+ [12] and also appears to have direct effects of its own to initiate exocytosis and cause changes leading to cell death. References 111Smith,

J.B., SD. Dwyer and Smith, L. (1989) Cadmium evokes inositol polyphosphate formation and calcium mo-

PI

bilization. J. Biol. Chem. 264, 7115-7118. Yamagami, K., Nishimura, S. and Sorimachi, M. (1994) Cd*+ and Co2+ at micromolar concentrations stimulate catecholamine secretion by increasing the cytosolic free

Ca2+ concentration in cat adrenal medulla cells. Brain Res. 646, 295-298. 131 Behra, R. and R. Gall (1991) Calcium/ calmodulin-dependent phosphorylation and the effect of cadmium in cultured fish cells. Comp. B&hem. Physiol., lOOC, 191-195. i41 Prozialeck, W.C. and R.R. Niewenhuis (1991) Cadmium (Cd2+) distrupts intercellular junctions and actin filaments in LLC-PK, cells. Toxicol. Appl. Pharmacol. 107, 81-97. [I Hinkle, P., Kinsella, P., Osterhoud, K. (1987) Cadmium uptake and toxicity via voltage-sensitive calcium channels. J. Biol. Chem. 262, 16333-16337. 161 Isom, G.E. and J.L Borowitz (1993) PC-12 cells. In: Methods in Toxicology, (Eds.) CA. Tyson and J.M. Frazier (Academic Press, New York) Vol. IA., pp.82-93. 171 Schanne, A.J., A.B. Kane, E.E. Young and J.L. Farber (1979) Calcium dependence of toxic cell death: A final common path way. Science, 206, 700-702. (81 Shukla, R. and Wakade, A. (1991) Functional aspects of calcium channels of splanchnic neurons and chromafftn cells of the rat adrenal medulla. J. Neurochem. 56,753-7X [91 Hart, D.T. and Borowitz, J.L. (1974) Adrenal catecholamine release by divalent mercury and cadmium. Archiv. Int. Pharmacodyn.

Ther. 209, 94-99.

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[IO] Lin, J., Sugimori, M., Llinas, R., McGuinness, T. and Greengard, P. (1990) Effects of synapsin I and calcium calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse. Proc. Natl. Acad. Sci. USA 87, 8257-8261. [I I] Niewenhuis, R.J. and W. C. Prozialeck (1987) Calmodulin inhibitors protect against cadmium induced testicular damage in mice. Biol. Reprod. 37, 127-133. [12] Shanbaky, LO., J.L. Borowitz, and W.V Kessler (1978) Mechanisms of cadmium- and barium-induced adrenal catecholamine release. Toxicol. Appl. Pharmacol. 44, 99-109. [I31 Cooper, G.P. and R.S. Manalis (1984) Cadmium: effects on transmitter release at the frog neuromuscular juntion, Eur. J. Phannacol. 99, 251-256. [I41 Guan, Y.Y., D.M.J. Quastel and D.A. Saint (1987) Multiple actions of cadmium on transmitter release at the neuromuscular juntion. Can. J. Physiol. Pharmacol. 65, 2131-2136. [15] Molgo, J., M. Pecot-Dechavassine and S. Thesleff (1989) Effects of cadmium on quanta1 transmitter release and ultrastructure of frog motor nerve endings. J. Neural Transm. 77, 79. [ 161 Fasitsas, C.D., S.E. Theocharis, D. Zoulas, S. Chrissimou and G. Deliconstantinos (1991)Time-dependent cadmiuminduced neurotoxicity in rat brain synaptosomal plasma membranes. Comp. B&hem. Physiol. IOOC, 271-275. [ 171 Hastings, L. and J. E. Evans (1991) Olfactory primary neurons as a route of entry for toxic agents into the CNS. Neurotoxicology 12, 707-714.

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[ 181 Gottofety, J. and H. Tjalve (1991)Axonal transport of cadmium in the olfactory nerve of the pike. Pharmacol. Toxicol. 69, 242-252. [I91 Pal, R., Nath, R. and Gill, K.D. (1993) Lipid peroxidation and antioxidant defense enzymes in various regions of adult rat brain after coexposure to cadmium and ethanol. Pharmacol. Toxicol. 73, 209-214. [20] Pal, R., Ravindra, N. and Gill, K. (1993) Influence of ethanol on cadmium accumulation and its impact on lipid peroxidation and membrane bound functional enzymes (Na+, K+, ATPase and acetylcholinesterase) in various regions of adult rat brain. Neurochem. Int. 23,451-458. [2l] Rosenberg, G., Estrada, E., Kelley, R. and Kornfield, M. (1993) Bacterial collagenase disrupts extracellular matrix and opens blood brain barrier in the rat. Neurosci. Lett. 160, 117-119. [22] Lo, W., Wolny, A., Timan, C., Shin, D. and Hinkle, G. (1993) Blood brain barrier permeability and the brain extracellular space in acute cerebral inflammation. J. Neural. Sci. 188, 188-193. [23] Tang, J., Xu, Z., Douglas, F., Rakhit, A., Melethil, S. (1993) Increased blood brain barrier permeability of amino acids in chronic hypertension. Life Sci. 53, PL417-420. [24] Ben-Shachar, D., Livne, E., Spanier, I., Zok, R. and Youdim, M., (1993) Iron modulates neuroleptic-induced effects related to the dopaminergic system. Isr. J. Med. Sci. 29, 587-592.