E L S E V I E R Chenuco-Blologlcal Interactions 93 (1994) 197-219

Chemico-Biological Interactions

Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and

astrocytes from rat brain

Juan Hidalgo *a, Agustina Garcia b, Anna M Oliva b, Mercedes Giralt a, Teresa Gasull a, Berta Gonzfilez a, Halina Milnerowicz a'l, Anne Wood c, Ian Bremner c

aDepartamento de Biologla Celular v Ftstologta bBzoqulmtca y Blologta Molecular and lnstttuto de Biologla Fundamental Vtcens Vtllar Palast, UmversMad Autonoma de Barcelona, Bellaterra,

Barcelona, Spare CThe Rowett Research lnstttute, Bucksburn, Aberdeen AB2 9SB, Scotland, UK

Received 7 January 1993, revision received 21 January 1994, accepted 26 January 1994

Abstract

The knowledge of bra in meta l lo th lonem (MT) regulation and especially of MT presence in specific cell types Is scarce Therefore, the effect of several well-known M T reducers, measured by ra&olmmunoassays using ant lbo&es that cross-react with MT-I and MT-II or specific for MT-I and which do not cross-react with human growth inhibi tory factor (GIF or MT-III), has been stu&ed m primary cultures of neurons or astrocytes obtained from rat cerebrum MT-I levels m ghal cells were abou t ten times higher than those m neuronal cells (538 ± 194 vs 49 ± 16 pg MT-I/t~g protein, mean ± S D from three separate cell preparat ions) Increas- Ing the concentra t ion of Zn in the bowne serum a lbumm (BSA)-contammg culture medmm up to 50/~M slgmficantly increased MT-I levels by up to 3 5-fold in neurons and 2 5-fold in astrocytes In contrast , Cu up to 50 # M increased MT-I levels m a saturable manner in both neurons (up to 5-fold) and astrocytes (up to 1 5-fold), the maximum effect occurring at 5 #M Cu. In general, the combina t ion of Zn and Cu further increased MT-I levels The effect of the metals on MT-I appeared to reflect metal uptake, since MT-I induction was less marked

* Corresponding author, Departamento de Blologla Celular y Flslologla, Umdad de Fls~ologla Ammal Facultad de Clenclas, Umversldad Autonoma de Barcelona, Bellaterra 08193, Barcelona. Spam Tel (3) 581 20 37, or (3) 581 16 64, Fax (3) 581 2000

1 Present address Me&cal Academy of Wroclaw, Department of Toxicology, Traugutta 57/59. Wroclaw 50-417, Poland

0009-2797/94/$07 00 © 1994 Elsevier Science Ireland Ltd All rights reserved SSDI 0009-2797(94)03276-E

198 J Hidalgo et al /Chem-Blol Interact 93 (1994) 197-219

when the BSA concentration in the medium was increased from 2 to 10 mg/ml Dex- amethasone increased MT-I levels in both neurons and astrocytes in vitro in a concentration- dependent manner Endotoxln, IL-l and IL-6 did not have a significant effect on ghal MT levels at the concentrations studied The administration of dexamethasone to rats increased MT-I levels m non-frontal cortex, cerebellum, pons + medulla, mldbraln and hlppocampus, but not m hypothalamus, frontal cortex and strlatum Endotoxln increased liver but not brain MT-I levels Immunocytochemlcal studies in adult rat brain preparations with a polyclonal antibody that cross-reacts with MT-I and MT-II indicated that immunostalnlng was always nuclear in ghal cells, whereas in neurons it was nuclear in the cerebral cortex, hlppocampus and the granular layer of the cerebellum, and nuclear plus cytoplasmic in Purklnje cells in the cerebellum, hypothalamlc nuclei and glgantocellular reticular nucleus in the brain stem Men- lnges, choroldal plexus, ependymal and endothehal cells were also MT-lmmunoreactlve

Keywords Metallothloneln, Zinc, Copper, Glucocortlcolds, Endotoxln, IL-I, IL-6, Neurons, Astrocytes, Brain

1. Introduction

Meta l lo th lone ln (MT) is a cysteme-rlch, low-molecular weight prote in that binds the essential metals zinc (Zn) and copper (Cu) Both metals are pr imary inducers o f MT synthesis, act ing at the t ranscr ip t iona l level through metal regula tory elements present in the M T gene, in mammals , M T gene t ranscr ip t ion is also mcreased by various hormones and growth factors, including glucocor t icolds [1-4]

Mos t a t tent ion has been centered on the occurrence o f M T in per lpherlc organs such as the liver and kidney, and little is known abou t its d is t r ibut ion, regulat ion and function in the brain, despi te the fact that both Zn and Cu are extremely impor- tant for normal bra in funct ion [5,6] The mechanisms by which these trace metals are t ranspor ted Into the bra in f rom the clrculat~on and accumula ted by bra in tissue are poor ly under s tood M T may p lay a central role in this regard M T prote in and m R N A levels are increased by ln t racerebrovent r lcu la r (lCV) but not per lpherlc ad- mlmst ra t lon o f Zn [7,8], whereas diets deficient in Zn decrease bra in M T levels [9], suggesting a role for this metal in the regula t ion o f bra in M T However , little is known abou t the role o f Cu on bra in M T regula t ion or o f the specific bra in cells (l e , neuron or ghal cells) in which M T is synthesized in normal (basal) condi t ions and m response to Zn and Cu exposure Indeed, the effect o f Zn (and/or Cu) in VlVO might even be indirect , since both the lCV admin is t ra t ion o f Zn and feeding a Zn- deficient diet might cause a stress-response, which can lead to bra in M T induct ion

[10,111 Therefore, the a im of the present work was to s tudy the d is t r ibut ion o f MT-I in

neuronal and as t rocyte-enr lched p r imary cultures from rat cerebrum and the regula- t ion o f MT levels by Zn, Cu and glucocort lcolds , individual ly or in combina t ion To know whether the differences in the MT-I levels between neurons and astrocytes observed m cul tured cells were comparab l e with the in VlVO si tuat ion, p r ehmmary ~mmunocytochemlcal studies were also under taken The dis t r ibut ion of MT-I in spe-

J Hidalgo et al /Chem-Bzol Interact 93 (1994) 197-219 199

cific brain areas and the effect thereon of perlphencally administered dexamethasone were measured by radIoimmunoassay to gain some insight into the role of glucocor- tIcolds on brain MT-I regulation in VlVO

2. Materials and methods

2 1 Ammals Male adult Sprague-Dawley rats were used for the m wvo and for the m wtro

hepatocyte studies For the m vitro neuron and gha stu&es, newborn (w~thm 24 h after birth) or 16-day-old embryos of either sex (about 50% each) were used follow- mg established procedures (see below) Ammals were maintained under standard condmons (lights on from 07 00 to 19 00 h, temperature 22°C, food and water pro- vided ad hbltum). Adults (3 months old), caged m groups of four, remained under those condmons for at least 1 week before the start of the experiments Zn and Cu content of the food was 95 and 35 mg/kg, respectwely The experimental protocols were approved by the appropriate mstltUtlonal review committee of the Autono- mous Umverslty of Barcelona and meet the European gmdehnes for ammal research actwmes

2 2 Matertals All chemicals were reagent grade Dexamethasone, zinc and copper were pur-

chased from Merck (Darmstadt, FRG) Cortlcosterone and endotoxin (hpopolysac- chande from Eschertchta cob 0127 B8) were purchased from Sigma (St Lores, MO, USA) Recombinant human xnterleukin-1 ct (IL-1) and interleukln-6 were a generous gift of Hoffman-La Roche (Nutley, N J, USA) and Genetics Institute (Cambridge, MA, USA), respectively Cell culture matermls were from Slgrna, Merck, Boehringer Mannheim (Mannheim, FRG), Glbco (Pmsley, Scotland) or Blowhlttaker (Walkers- ville, Maryland, USA) Laboratory food was from Panlab (Barcelona, Spain)

2 3 Prtmary cultures Astrocyte-enrlched and neuronal primary cultures were prepared from the brain

hemispheres of newborn rats (within the first 24 h) and 16-day-old embryos, respec- tively, following the method of Loffner et al [12] as previously described [13] Brief- ly, for astrocyte cultures, tissue was &ssooated into cells by successwe passages through nylon cloths of 211- and 135-/~m mesh Bram cells were suspended in 90% Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), 20 units/ml pemcdhn and 20/~g/ml streptomycin (0 6 × 106 viable cells/ml) and then seeded onto 35-mm diameter plastic Petn dishes (2 ml) and Incubated at 37°C m a humidified atmosphere of 90% air/10% CO 2 Medium was changed once a week, and confluent monolayers were used at 13-14 days m culture because at this time cells are confluent and the number of contammatIng cells is low

For neuronal cultures, tissue was passed through nylon cloths of 135- and 22-#m mesh and cells were suspended in 90% DMEM, 10% horse serum, 20 units/ml pem- cilhn, and 20 ~g/ml streptomycin Two mllhhters of the cell suspension (0 6 × 106 viable cells/ml) were seeded onto 35-mm &ameter plastic Petn &shes previously

200 J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219

coated with poly-L-ot-ornlthlne (0.01% wt/vol) After 1 day in culture the initial cul- ture medium was replaced by serum-free gha-conditioned medium containing Insulin (5 ttg/ml), transferrin (100/xg/ml), putrescine (100/~M), progesterone (20 nM), and Na2SeO3 (30 nM) Two days later cytosine arablnoslde (5 #M final conc) was added and after 24 h the medium was replaced by hormone-supplemented condition- ed medium Neuronal cultures were incubated in the same way as the astrocytes and were used at 11-12 days in culture because at this time neurons are well differen- tiated and the number of contaminating cells is low

Isolated liver parenchymal cells were prepared from adult male Sprague-Dawley rats by using the three-step perfuslon technique of Garvey and Heil [14] as modified [15,16] The collagenase was perfused in RPMI 1640 The hepatocytes were washed three times with Dulbecco minimum Eagle medium (DMEM F-12) containing 20 mmol/l HEPES, 14 mmol/l NaHCO3, 100/zg/ml penicillin, 60/~g/ml streptomycin, and 50/~g/ml gentamycin to remove non-parenchymal cells Parenchymal cells were finally suspended (l 1 x 10 6 cells/ml) in DMEM F-12 medium supplemented with 20 mmol/l HEPES, 14 mmol/1 NaHCO3, 100 #g/ml penicillin, 60 /~g/ml strep- tomycin, 50 ~g/ml gentamycin, 1 #g/ml insulin and 10% fetal calf serum Three milliliters of this suspension were plpetted onto 60-mm plastic culture dishes, which were previously coated with collagen following the instructions of the manufacturer Viable cells were allowed to attach selectively for a 4-h incubation period at 37°C in a 5% CO2, HEO-Saturated atmosphere The medium was then removed and the cells were washed once with 3 ml of DMEM F-12 supplemented with 20 mmol/1 HEPES, 14 mmol/l NaHCO3, 100/zg/ml penicillin, 60/~g/ml streptomycin, and 50 /~g/ml gentamycin Three mllhhters of fresh DMEM F-12 supplemented with 20 mmol/1 HEPES, 14 mmol/l NaHCO3, 100/~g/ml penicillin, 60/~g/ml streptomycin, 50/zg/ml gentamycin, 10/~M ZnSO4 to approximate a physiological plasma Zn con- centration and 2 mg/ml bovine serum albumin (BSA), were added to the dishes. The hepatocytes were then incubated for approximately 20 h at 37°C in a 5% CO 2, HaO- saturated atmosphere

2 4 Effect o f Z n , Cu, glucocorttcotds, endotoxtn, IL-1 and IL-6 on M T levels in cells in culture

Experiments were started after the culture periods stated above For the experi- mental treatments in astrocyte cultures, the medium was removed, the monolayers washed with 2 ml DMEM to remove FBS, and 2 ml of fresh DMEM supplemented with 2 mg/ml bovine serum albumin (BSA) (unless otherwise stated) were added In neuronal cultures, the medium was removed and substituted by the same culture me- dram without antibiotics and supplemented with 2 mg/ml BSA (unless otherwise stated) Zn, Cu, dexamethasone or corticosterone, endotoxin, IL-1 and IL-6 were then added in a volume of 50/~1 to reach the desired final concentrations (see Figures and Tables) and the cells incubated for 24 h The medium was then removed and centrifuged (3000 x g, 15 mln) at 4°C to eliminate detached cells, and the superna- tant was stored at -20°C until required for MT assay The cells were gently washed with 1 ml of 10 mM Tris-HCl (pH 8 2), scraped with a spatula m 2 ml of the same buffer, and sonicated for 20 s at 4°C After centrlfugation (3000 × g, 15 mln, 4°C),

J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219

the supernatant was stored at -20°C until reqmred for MT assay same procedure was followed for cultured hepatocytes.

201

Essentmlly the

2 6 Effect o f endotoxm m vtvo Rats were injected 1 p with endotoxm (1 mg/kg) and kdled 8 h later Livers and

brains were dissected and stored at -85°C

2 7 lmmunocytochemtstry Immunohlstochemlcal staining for MT was carried out by the streptavldm-blotm

techmque After anesthesia with sodium pentobarbital, three rats (4-month-old) were perfused for 20 mm with 4% paraformaldehyde m 0 1 M cacodylate buffer (pH 7 4) containing 5% sucrose The brains were then removed and samples were dehydrated and embedded m paraffin Frontal plane sections (10/~M thick) were obtained and mounted on glass shdes covered with a thin polylyslne layer

Before lmmunostammg procedures, the sections were deparaffimzed, rehydrated in ethanol and rinsed m 0.05 M Trls-buffered sahne (TBS) (pH 7.4) containing 1% Triton X-100 The shce-mounted sections were then incubated for 30 mln with 10% fetal calf serum (FCS) dduted in 0 05 M TBS (pH 7 4) to reduce non-speofic stare- rag, whereupon they were incubated overmght at 4°C with rabbit antibody to MT, dduted 1:50 m 10% FCS Th~s polyclonal antibody cross-reacts equally well with MT-I and MT-II [17], but only shghtly with human MT-III or growth inhibitory fac- tor, GIF ([5912 Uchlda, Y , unpubhshed observattons) Following rmsmg with TBS containing 1% Triton X-100, the sections were incubated with a secondary antibody blotmylated (Amersham RPN 1004) for 60 mm, treated with 0 2% H202 m 100% methanol for 30 mm to block endogenous peroxldase, rinsed again in TBS with 1% Triton X-100, and incubated with peroxldase-conjugated streptawdln (Merch 20049) for 45 mm. After rinsing in TBS, the peroxldase was reacted with 0 16% H202 for 5 mm using dmmmobenzldme (DAB) as chromogen After rlnsmg in TBS, sections were counterstalned with Harns-hematoxyhn, dehydrated m ethanol, cleared in xy- lene and covershpped in Dammar resin

The speoficity of the lmmunohIstochemlcal staining was controlled by (a) pro- cessmg brain sections in non-~mmune rabbit serum without the primary antibody, and (b) by preabsorblng the primary antibody with rat hver MT before staining was carried out As control t~ssue, liver sections were also used

2 The following reference was added m proof [59] T Gasull, M Glralt, J Hernandez, P Martmez, I Bremner and J Hidalgo, Regulation of metallothloneln concentrations m rat brain effect of gluco- cortlcoIds, zinc, copper, and endotoxm, Am J Physlol , 266 (1994) E760-E767

2 5 Effect o f dexamethasone m vzvo Rats were rejected i p. with 2 mg/ml dexamethasone (dissolved m sahne) at 09 00

h or with sahne alone (n = 7) They were kdled after 8 h by decapitation and the brains and livers immediately removed and stored at -85°C Brains were &ssected into the following 8 areas' frontal cortex, remaining cortex, hlppocampus, hypothal- amus, cerebellum, stnatum, pons+ medulla oblongata and m~dbram, and stored at -85°C For MT assay, brain area samples were prepared as previously described Ill].

202 J HMalgo et al /Chem -Btol Interact 93 (1994) 197-219

2 8 Assays

Cellular, extracellular and tissue MT-I levels were measured by a highly specific and sensitive RIA [18], this antxbody does not have cross-reactwlty with either MT- II or human growth inhibitory factor, MT-III (see below) In some experiments, both MT-I and MT-II were analyzed with an alternatwe RIA [17] Cellular protein content was determined as described by Bradford [19] with BSA as standard At- tempts were made to measure Zn and Cu levels m the cells by ICP-MS, but these were unsuccessful because of the small amount of the metals Cell cultures were assayed m duphcate or tnphcate for each experimental condmon, and m most cases the experiments were repeated 2 or 3 times with &fferent culture preparations When the experiments were done only once, they were for confirmatory purposes Results, expressed as percent of control cells, were analyzed with the Student t-test, the Mann-Whitney U-test, one- or two-way ANOVA where appropriate when two or more cell preparations were carried out When only one experiment was performed the mean values are shown but they have not been statistically examined When necessary, the data were subjected to logarithmic transformation to achieve homogeneity of variance

3. Resu l t s

In most experiments, MT-I levels were measured by ra&olmmunoassay using an antibody that does not cross-react wzth MT-II Fig. 1 shows that no cross-reactwlty of this antibody was observed with human growth inhibitory factor (MT-III), an MT lsoform only expressed in the brain, suggesting that the present results mostly reflect MT-I levels. Basal MT-I levels m cultured astrocytes and neurons as described m

6] Z D 0

100

80

60

40

20

0 50

i i i l l i J i i i i l l ~ i i L i i i i i I X ~ - ~ - ' - ~ ) l

1 O0 1000 10000

L O G C O M P E T I T O R (pg)

© MT-1 - - ~ - GIF

Fig 1 Cross-reacUwty of human growth inhibitory fac.tor (GIF or MT-III) with a polyclonal antibody raised against rat MT-1 The concentrations of MT-I and GIF are as stated

J Htdalgo et a l /Chem-Btol Interact 93 (1994) 197-219 203

Mater ia ls and methods , were subs tant ia l ly different, since they were abou t 10 times htgher m glial cells than m neuronal cells (538 ± 194 vs. 49 ± 16 pg MT//~g protein, mean ± S . D , f rom 3 independent cel lular p repara t ions ) (P < 0 001) This was sup- por ted m vlvo by lmmunohts tochemlca l s tu&es m adul t rat bra ins (Fig 2), using an anUbody developed agamst ra t MT-I I , which cross-reacts equal ly well with MT-I and MT- I I [17] but that only has a weak c ross - reacuwty with human G I F (Gasul l et al. [59]). The speclfictty o f the immunos t aming for M T was es tabl ished by the ab- sence o f reactivi ty m cont ro l sections incubated wi thout a p r imary an t ibody Stain-

mg with a p r imary an t ibody previously p reabso rbed with M T (8 #g/ml) also resulted in a substant ia l reduct ion in s ta ining In addi t ion , hver sections showed the expected s t rong cytoplasmic immunos t a lnmg

In bra in pa renchyma (Fig 2a), the MT-pos l t tve cells consisted mamly o f neurons and gha cells. The degree o f i m m u n o s t a m m g was, however, very vartable, some cells showing a clear lmmunoreac twe produc t , whereas others showed only weak lm- munoreacuwty Fur the rmore , m all areas s tudied it was posstble to identify both neuronal and ghal non - lmmunoreac twe cells (blue) a long with tmmunoreac t lve cells (brown) The degree of t m m u n o s t a m m g was general ly higher in ghal than in neuro- nal cells.

Immunoreac twl ty m MT-pos i t tve neurons was usual ly located in the nucleus, a l though in par t icu lar areas the cy top lasm was also s tained Thus, in the cerebral cortex, h lppocampus and the g ranu la r layer o f the cerebellum, only the nuclei exhibi ted M T lmmunoreac t lwty (Fig 2b) In contrast , Purkmje cells in the cerebellum, neurons o f the g lgantocel lu lar re t icular nucleus o f the bra in stem and the two nuclei o f the hypo tha l amus s tu&ed, vent romedia l and arcuate, showed MT-lmmunoreacUvi ty m bo th nuclei and cy top lasm (Figs 2c,d)

M T - p o s m v e neurons were observed scat tered in all areas s tu&ed, wi thout any ap- parent pat tern o f & s t n b u t l o n However , m the cerebral cor tex MT-post t tve neurons were absent m layers 3 and 5, pyramtda l cells always a p p e a r m g unlabel led In con- trast, all neurons o f the g lgantocel lu lar ret tcular nucleus of the bra in stem were labelled

Fig 2 P.

Fig 2 (a) Cerebral cortex MT-posltlVe neuronal and ghal cells (arrows) are scattered in the brain paren- chyma Note that MT lmmunoreactwlty is also related to meninges (M) × 386 (b) Cerebellar cortex Main neurons in the granular layer (GL) showed MT-lmmunoreactlvlty related to nuclei Arrow points a Purklnje cell with noticeable MT-lmmunoreactlwty m the nucleus Note the presence of positive cells in the molecular layer (ML) x618 (c) Cerebellar cortex The mlcrograph shows presence of non- Immunoreactwe Purklnje cells (arrowheads), as well as Purklnje cells with dlscermble lmmunoreactwe product in the nucleus and cytoplasm simultaneously (arrows) ML. molecular layer GL, granular layer × 618 (d) Secuon of the Glgantocellular reticular nucleus in the brain stem showing a group of MT- posmve neurons with lmmunoreactlvlty located in the nucleus and cytoplasm simultaneously × 618 (e) Corpus callosum There was evidence of the presence of MT-posmve ghal cells (arrows) in the rows of ohgodendrocytes among the myellnated fibers Note that endothelial cells appeared also stained (ar- rowheads) × 618 (f) Mlcrograph corresponding to ependymal layer in third ventricle where tt was possi- ble to observe immunoreactwe cells (thin arrows) x 386 (g) Choroldal plexus of the lateral ventricle MT Immunoreactwlty was found in association with nuclei of the cells (arrows) Note the presence of non- lmmunoreactlve cells (arrowheads) ×618

204 J Hidalgo et al /Chem-Btol Interact 93 (1994) 197-219

J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219 205

206 J Htdalgo et al /Chem -Blol Interact 93 (1994) 197-219

N e u r o n s

% vs Control 1600

1400

1200

lOOO

s o o " k T

600 * m [] D

400 E - ±.

2001 / ~

0 0 10 20

E3

/,,

I I I H

30 40 50 p M

o Zn - ~ - Cu [] Z n + C u

G l i a

4 0 0

300

2 0 0

100

% vs Control

[]

[ ]

/ - -

0 ' ' ' ' ' p M 0 10 20 30 40 50

o Zn - ~ - Cu [] Z n + C u

Fig 3 Effect of Zn, Cu or Zn + Cu on MT-I levels m primary cultures enriched m neurons (neurons) or astrocytes (gha) The cells were incubated for 24 h with the specified concentrations of the metals m the presence of 2 mg BSA/ml medmm When both metals are present, the concentrations stated are of each metal MT-1 levels are expressed as percentage of MT-I vs control cells (incubated w]th no metals m the medmm) The results are mean ± S E from two to four independent cellular preparattons, m each cellular preparation, 2 -3 rephcates were done of each experimental point *P < 0 05 vs control cells (Duncan test) For further statistical comparisons, see Results

J HMalgo et al /Chem-Bzol Interact 93 (1994) 197-219 207

MT-lmmunoreactlvlty m ghal cells was found exclusively m the nucleus As with neurons, MT-posxtlve gllal cells were scattered in both gray (Fig 2a) and white matter among the rows of ohgodendrocytes (Fig 2e) Ependymal (Fig 20, endothe- hal (Fig 2e) and choroldal plexus (Fig 2g) cells also showed MT lmmunoreactlvlty In some experiments with human brain, the lmmunostalnlng of this polyclonal anu- body was compared with that of the monoclonal antibody E9 [20,21], the two ant~- bo&es giving essentially the same results (data not shown)

Fig 3 shows the concentrations of MT-I m neuron and gllal cells incubated for 24 h in the presence of 2 mg BSA/ml and 0, 0 5, 5, 25 or 50/zM Zn and/or the same concentrations of Cu For cultured neurons, one-way ANOVA for each metal m- dlcated that Zn (P < 0 001), Cu (P < 0 05) and Zn + Cu (P < 0 001) significantly increased MT-I levels Post-hoc comparisons are shown m the figure When com- pared by two-way ANOVA, MT-I levels were significantly higher in Cu-treated cells than in Zn-treated cells (P < 0 005) Cells incubated with Zn + Cu had higher MT-I levels than Zn- (P < 0 001) or Cu-treated cells (P < 0 05)

In cultured ghal cells, Zn (P < 0 01) and Zn + Cu (P < 0 01) significantly increas- ed MT-I levels The effect of Cu did not reach statistical slgmficance (P = 0 067) Post-hoc comparisons are shown in the figure There were no statistically significant differences between MT-I concentrations in cells incubated w~th Zn compared with those incubated with Cu or Zn + Cu In contrast, MT-I levels m gllal cells mcubated with Zn + Cu were s~gnxficantly h~gher than in cells mcubated w~th Cu alone (P < o 05)

Table 1 shows the effect of increasing the concentratmn of BSA m the medmm from 2 to 10 m~ml on MT-I concentrations m cells exposed to 50 #M Zn, Cu or Zn + Cu As expected, neurons and ghal cells incubated m the presence of 10 mg BSA/ml showed a greatly reduced MT-I response compared to those cultured w~th 2 mg/ml

F~g 4 shows the effect of dexamethasone on MT-I levels m cultured neurons and gha Dexamethasone s~gnlficantly increased MT levels m a concentration-dependent

Table 1

Effect of &fferent BSA concentratmns on the effect on MT-1 concentrattons of Zn, Cu or Zn + C u m primary cultures enriched m neurons (neurons) and astrocytes (gha)

Neurons Gha

2 mg/ml BSA 10 mg/ml BSA 2 mg/ml BSA 10 mg/ml BSA (2-3) (1) (2-3) (l)

Control 100 + 13 5 100 99 7 ± 20 8 100 50/zM Zn 363 ± 43* 102 247 ± 37* 110 50/zM Cu 469 ± 123" 98 170 m 29 121 50 #M Zn + Cu 1204 ± 364* 164 309 ± 61" 151

The cells were incubated for 24 h with the stated metal and BSA concentrations Results are the mean ± S E , expressed as percentage of MT-I vs control cells, number of separate cell preparations m parentheses For 10 mg/ml BSA a stogie experiment was performed and only the mean of the rephcates is shown *P < 0 05 vs control cells (Duncan test)

208 J HMalgo et al I Chem -Bwl Interact 93 (1994) 197-219

400

320

240

160

80

% vs Control

t ~t ~t

. T

T X _ - . . . . - /

/

/ /

/ J " / 'k / /

o -'8 ! r ' '-s .4 M CONTROL 10 M 10 M 10"6M 10 M 10

o N E U R O N S - - ~ - - GLIA

Fig 4 Effect of dexamethasone on MT-I levels of cultured neurons (neurons) and astrocytes (gila) The cells were incubated for 24 h with increasing hormone concentrations (10 - s - 10 -4 M) in the presence of 2 mg/ml BSA Results are mean ± S E from two independent cellular preparatmns MT-I levels are ex- pressed as percentage of MT-I vs control cells For other details, see Legend of Fig 1 *P < 0 05 vs control cells (Duncan test)

manner m both neurons (P < 0.05) and gha (P < 0 05). Post-hoc comparisons between means are shown (Fig. 4).

Table 2 shows the effect o f the natural glucocorttcold, cortlcosterone, on MT-I or MT-I + II levels In general, the results are m agreement with the effect o f dexa- methasone

Table 2 Effect of the natural glucocorncold cortlcosterone on MT levels m primary cultures enriched in neurons

(neurons) and astrocytes (gha)

Neurons Gha

Exp 1 Exp 2 Exp 1 Exp 2+3 (MT-I) (MT-I + II) (MT-I) (MT-I + II)

Ethanol 100 100 100 99 8 ± 0 14 10 -8 M 106 - - 70 - - 10 -7 M 128 99 94 - - 10 -6 M 141 - - 120 296 ± 111 10 -5 M 132 90 5 92 - - 10 -4 M 146 247 106 602 ± 59*

The cells were incubated for 24 h with the stated corUcosterone concentrations (dissolved first in ethanol) m the presence of 2 mg/ml BSA Results are the mean ± S E , expressed as percentage of MT vs control cells If a single experiment was performed only the mean of the rephcates is shown *P < 0 05 vs cells

incubated with ethanol alone (Duncan test)

J Htdalgo et a l /Chem -Bwl Interact 93 (1994) 197-219 209

Table 3 Effect of l/~M dexamethasone alone or combined with 5/~M Zn, Cu or Zn + Cu on MT levels of cultured neurons (neurons) and astrocytes (gha)

Neurons Gha

Exp 1 Exp 2 Exp 1 Exp 2 (MT-I) (MT-I + II) (MT-1) (MT-I + II)

Control 100 100 100 100 Zn 172 176 91 72 Cu 408 583 147 110 Zn + Cu 371 581 164 92 Dexamethasone 313 847 359 159 Dexamethasone + Zn 460 1264 380 180 Dexamethasone + Cu 1230 1544 549 227 Dexamethasone + Zn + Cu 1374 1302 587 257

The cells were incubated for 24 h with the hormone and the metals in the presence of 2 mg/ml BSA Re- suits show the mean of the rephcates of two separate cell preparations, and are MT levels expressed as percentages vs control cells If the data are pooled for statlsucal purposes, two-way ANOVA m&cated that both dexamethasone and metals had a slgmficant effect on MT levels m neurons (P < 0 001 m both cases), whereas only dexamethasone had a slgmficant effect on MT levels m ghal cells (P < 0 001)

1 0 0 0

8 0 0

6 0 0

Hepatocytes

% v s C o n t r o l

/ /

4 0 0 ~ / /

_ T / / /

2OO -~ / J~ -

0 I I

0 1 0 2 0

6

- 4

3

c- 2

J i i 0

3o 40 5o pM Zn Cu Zn Cu - D E X + D E X

o Z n - - ~ - - C u

Fig 5 Effect of Zn or Cu on metallothlonem levels in primary cultures of hepatocytes The cells were incubated for 24 h with the specified concentrations of the metals m the presence of 2 mg BSA/ml medmm Those cells incubated with Cu were concomitantly incubated with 10 ~.M Zn MT levels are expressed as percentage of MT vs cells incubated w~th 10 #M Zn The results are mean ± S E from two to four independent cellular preparations, tn each cellular preparation, 2-4 rephcates were done of each experi- mental point *P < 0 05 vs control cells (Duncan test) On the right is also shown the effect of dex- amethasone on Zn and Cu reduction of MT in one hepatocyte preparation, in this case, the mean of the rephcates is shown

210 J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219

Table 3 shows the effect of 5 t~M Zn, Cu or Zn + Cu on neuronal and ghal cells in the absence or presence of 10 -6 M dexamethasone This experiment was carried out twice, only MT-I was measured in the first (as in the previous experiments), whereas both MT-I and MT-II levels were measured in the second If the results from both experiments are pooled for statistical purposes, two-way ANOVA with metals and dexamethasone as the mare factors indicated that both factors had a sig- nificant effect on MT levels m cultured neurons (P < 0 001), whereas only dex- amethasone had a slgmficant (P < 0001) effect m ghal cells The interaction between the two factors was not significant m either neurons or gha, indicating that there was no synergy between the hormone and the metals

For comparison, the effect of zmc, copper and glucocortlcoids is also shown In primary cultures of rat hepatocytes (Fig. 5) As expected, both zinc and copper in- creased MT levels m a concentration-dependent manner (P < 0 001) Post-hoc com- parisons are shown m F]g 5 The effect of dexamethasone lS also shown for one cell preparation

The above effects of glucocortlcolds on MT levels in cultured neurons and astro- cytes suggested that these hormones might be revolved In brain MT-I regulation in wvo An experiment was therefore performed to estabhsh whether the synthetic glucocortlco]d dexamethasone can influence MT-I levels in specific brain areas As expected, dexamethasone slgmficantly increased (P < 0 01) liver MT levels 8 h after

4

Brain metallothionein-I

O

O3

O3

3

2

1

5

0 FR C O R C O R M+P MIDB STRL HlPP H Y P O CERE

SALINE ~ DEX

F]g 6 Effect of dexamethasone on MT-I levels of spec]fic brain areas Dexamethasone (2 mg/kg) was re- jec ted mtrapentoneally and the rats were kdled 8 h later Results are mean 4- S E (n = 7) *P < 0 05 vs sahne rats (Student t-test) For further statistical comparisons, see Results Abbreviations Fr Cor , frontal cortex, Cor, remaining cortex, M + P, medu l l a + pons, Mldb, m~dbram, Stn, stnatum, Hlpp, hlppocam- pus, Hypo, hypothalamus, Cere, cerebellum

J HMalgo et al /Chem-Btol Interact 93 (1994)197-219

Table 4 Effect of endotoxm, IL-1 and IL-6 on MT levels of primary cultures enriched in astrocytes

211

Control 99 8 4- 0 14 Endotoxm (100 t~g/ml) 161 ± 42 IL-1 (100 umts/ml) 133 ± 74 IL-6 (10 umts/ml) 167 4- 76

The cells were incubated for 24 h with the stated concentrations of endotoxm, recombinant human I L-I or IL-6 Results are mean 4- S E of two independent cell preparations MT levels are expressed as a percentage vs control cells

its administrat ion (8 7 4- 1 52 and 19 39 4- 2 53 for sahne and dexamethasone-

treated rats, respectively) Fig 6 shows the effect o f dexamethasone on MT-I levels

m 8 specific brain areas o f the same ammals In basal condmons , MT-I levels were

higher m strmtum and hypotha lamus (P at least < 0 05) than m the remaining brain

areas, except for MT-I levels m frontal cortex which were not significantly dtfferent

from those o f the hypothalamus The h lppocampus and the frontal cortex had rater-

mediate MT-I levels (P < 0 05) The non-fronta l cortex, medul la plus pons, mid-

brain and cerebellum had the lower MT-I levels, with the non-frontal cortex having

significantly higher MT-I levels than the other 3 areas The administrat ion of

dexamethasone xncreased MT-I levels slgmficantly m the non-frontal cortex,

medulla + p o n s , mldbraln, h lppocampus and cerebel lum (P at least < 0 05), with the

greatest response occurr ing in the cerebel lum

Table 4 shows the effect o f endotoxln, IL-I and IL-6 on ghal M T levels None of

them increased M T levels significantly at the concentra t ions stu&ed Th~s was m

agreement w~th the results obtained m wvo m rats injected w~th endotoxln (Table

5), which dtd not show mcreased brain M T levels m spite o f mcreased hver MT

levels

4. Discuss ion

4 1 Cells tn cul ture

The results indicate that there are similarities but also substantial &fferences in

the regulation o f M T - | in both cell types MT-I levels were found to be ten times

Table 5 Effect of endotoxm on brain and bver MT-I levels m the rat (t~g/g tissue)

Sahne Endotoxm

Cortex 1 69 + 0 07 1 84 ± 0 07 Cerebellum 1 23 ± 0 10 1 52 ± 0 11 Lwer 98 ± 2 2 408 ± 15'

Results are mean ± S E (n = 5-8) Rats were mjected with endotoxm (1 mg/kg) and kdled 8 h later *P < 005vs sahnerats

212 J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219

higher m cultured astrocyte-enrlched (at least 90%) cells than m neuronal cells Zn (0 5-50 #M) significantly increased MT-I levels m both the neuronal and astrocyte- enriched cultures The MT-I elevation was modest, but similar to that observed m cultured hepatocytes incubated with Zn under the same conditions (present report, [22]) At this concentration (30/~M), BSA should act as a Zn binding protein [23], hmltlng therefore the metal uptake We have also to bear m mind that non-BSA Zn can mteract w~th hlstldme m the medmm, which might interfere with metal uptake, as was found m synaptosomes [24] Unfortunately, it is not known how much free Zn was present at each extracellular metal concentration, and m which form Zn was taken up by the cells under our experimental conditions However, the results strongly suggest that the changes observed in MT-I levels are a consequence of &f- ferences m metal uptake, smce when cells were cultured in the presence of 10 mg BSA/ml medium instead of 2 mg/ml (therefore decreasing free Zn levels), the effect of Zn was abohshed An enhanced Zn uptake might, m principle, Increase MT-I syn- thesis, since this metal is a direct reducer of the MT gene [2,4] Unfortunately we were not able to measure mtracellular Zn levels because of the small s~ze of the sam- ple Howell et al [25] have demonstrated the existence of two Zn transport systems operating in parallel m hlppocampal shces, a high-affinity system (K m = 12 /~M, Vmax = 5 8 ng Zn/mg per h) and a low-affimty system (Km = 402/~M, Vma x = 113 ng Zn/mg per h) As these uptake processes were observed in the absence of physiological chelators (BSA, hlstldlne), we suggest that under our experimental conditions Zn uptake ~s operating by the high-affinity, low-capacity system

S~mdar increases m MT concentrations were found m human neuroblastoma IMR-32 cells incubated m the presence of 100/~M Zn and 10% fetal calf serum [26] In contrast, MT levels m rat h~ppocampal neurons m primary cultures were mcreas- ed by 0 01, 0 1 and 1 /xM Zn but not by higher Zn levels [27] However, these cells were incubated m the absence of serum or albumin, and thus much of the Zn would have been m free form, the metal uptake might therefore have been higher than in our experimental conditions The lack of induction of MT by 10 or 100 #M Zn sug- gests mcreased toxicity of the free, non-protem bound metal to the neurons [26]

As with Zn, the addition of Cu to the culture medm slgmficantly increased MT-I levels It is hkely that this reduction reflected Cu uptake by the cells, since increasing the BSA concentration abolished the effect of Cu Unfortunately, we could not mea- sure Cu levels m the cells However, Cu reduced a different pattern of response com- pared to Zn, insofar as it increased MT-I levels more in neuronal (5-fold) than in ghal (1 5-fold) cells, and exerted its maximum effect at 5 txM This suggests the existence of a saturable uptake process in both cellular populations There have been reports of two hgand (hlstldme)-dependent saturable processes for Cu accumulation m rat hypothalamlc tissue shces, wz a hlgh-affimty, low-capacity process (Km = 6~tM, Vmax = 23 pmol/mln per mg) and a low-affinity, high-capacity process (Km = 40/~M, Vma x = 425 pmol/mm per mg) [281 The hgand specificity for uptake of complexed Cu is a function of Cu concentration and Cu hgand molar ratio [291 Other amino acids m the culture medmm can also facdltate Cu uptake by hypothalamlc shces to various degrees [28,29], although preferentml chelation of the Cu by BSA may block their effects [28,30] It is &fficult to determine which uptake

J HMalgo et al/Chem -Btol Interact 93 (1994) 197-219 213

process is involved in the present study, since it is not known If results from hypothalamlc and other brain tissue slices [28,30] can be extrapolated to cultured neuronal or glial cells I f they can, Cu uptake by these cells may operate by the high- affinity, low-capacity system, since it is saturated above 5/~M Cu

When both Zn and Cu were present in the medium, different responses were again seen in the cultured neurons and gha, in the former, there was an additive effect between both metals, whereas in the latter, the effect was less than additive These results probably also reflect metal uptake processes, as the presence of 10 mg/ml BSA In the medium again greatly decreased the effect of metals on MT-I levels, and suggest that ghal cells incorporate Zn and Cu less efficiently than neuronal cells Alternatively, MT-I may be less inducible by metals in ghal cells

Dexamethasone, a well-known MT inducer [2,4], also increased MT-I levels in both neurons and gha in a concentration-dependent manner, even in the presence of BSA and with no metals added in the culture media, indicating that glucocortlc- olds may potentially regulate MT-I levels in the brain The effect of cortlcosterone, the natural glucocortlcoid in the rat, was much less than that of dexamethasone but followed the same trend This is consistent with the well-known greater potency of dexamethasone. No significant effect of dexamethasone on MT-I accumulation was found in human neuroblastoma IMR-32 cells [26], which could reflect a loss of response of this cancerous cell line regarding MT

As dexamethasone potentiates the uptake of Zn and to some extent of Cu in cul- tured hepatocytes [31-33], cultured neurons and gha were exposed to l /zM dex- amethasone in the absence or the presence of 5 #M Zn, Cu or Zn + Cu The combined effect of dexamethasone and metals appears to be additive, tending to be synergistic in neurons, but to be less than additive in gha, suggesting that glucocor- tIcOlds and metals act through independent mechanisms as is actually known to hap- pen in peripheric tissues

Substantial amounts of MT-I were observed in the culture media after the dif- ferent treatments, especially in neurons (not shown) However, the results were not consistent between experiments, and therefore the putative MT secretion by either neurons or gha needs further characterization

4 2 Immunocytochemtstry

MT-lmmunoreactlvIty was detected in both neuronal and gllal (presumably astro- cytes) cells in most brain areas The presence of MT in astrocytes and ItS nuclear and perinuclear localization has been demonstrated in rats and mice [34-37], in Macaca fasctcularls [381 and in humans [21,39] However, the detection of MT in neurons with our polyclonal antibody in most brain areas has not been reported in previous studies with other antibodies Only Blaauwgeers et al [21], using the monoclonal antibody E9, have observed MT lmmunostalnlng in neurons of hypoglossal nucleus of medulla oblongata The reason for these discrepancies is unknown, but un- doubtedly it may have to do with the fact that MT levels are 10 times higher in astro- cytes than in neurons, at least in primary cultures of these cells, which could have precluded the detection of MT in neurons in previous studies In addition, several discrepancies are also evident in the literature available in other aspects Thus,

214 J Htdalgo et al /Chem -Btol Interact 93 (1994) 197-219

Nlshimura et al [37] did not find significant immunostainlng in normal adult rat brain, whereas Young et al [36] found strong lmmunostalnlng In ghal and epen- dymal cells, in contrast, Nakajima et al [40] found MT presence in ependymal but not in ghal cells In humans, Suzuki et al [39] found MT immunostalnlng in astro- cytes of grey and white matter, and in ependymal cells and endothelial cells of cho- roid plexus, whereas Blaauwgers et al [21] found MT presence only in astrocytes of grey matter It therefore seems clear that different antibodies are giving substan- tially different results in lmmunocytochemlcal studies In the present report, it ap- pears that given (a) the absence or reduction of the lmmunostalnlng with the appropriate controls, (b) the similarity of the response of the polyclonal antibody used compared with the monoclonal E9 in human brain (not shown), and, above all, (c) the detection of MT-I by RIA in primary cultures of neurons, the results support the normal presence of MT in neurons of adult rat brain in addition to astrocytes

Regardless of the brain area studied, MT appeared to be present exclusively in the nuclei of ghal cells However, ultrastructural studies need to be performed to con- firm this, since the cytoplasm of these cells is reduced, Nlshlmura et al [37] report the cytoplasmic presence of MT in these cells In the case of neurons, MT was pre- sent in the nucleus alone or in the nucleus plus cytoplasm, depending on the area studied The simultaneous occurrence of cells with positive or negative staining in most areas studied suggests that the induction of MT synthesis in individual neurons and ghal cells depends on their particular physiological state, with preferential nu- clear accumulation in some neuron types and in all (when increased) ghal (astrocyte) cells The fact that in some areas the neurons consistently contained MT in both the nucleus and the cytoplasm whereas in others MT was present only in the nucleus (see Results) suggests a functional difference between the neurons of different brain areas MT is usually considered to be a cytoplasmic protein but its transient presence in the nucleus of hepatic cells has been described during development [41] and after partial hepatectomy [42]

Other features of the immunocytochemlcal results are intriguing MT was absent from layers 3 and 5 of the cerebral cortex, whereas it was present m all neurons of the gigantocellular reticular nucleus in the brain stem, which again highlights possi- ble functional differences On the other hand, MT was clearly present in blood vessel endothelial cells and in ependymal cells, suggesting its involvement in metal (Zn, Cu) uptake/exchange processes between the brain and blood and/or the cerebrospinal fluid (CSF), respectively MT could also be important in the protection of the brain against toxic metals such as cadmium Nishimura et al [37] have recently reported MT induction by cadmium in both ependymal and endothelial cells The choroid plexus strongly accumulates administered cadmium, but no cadmium could be detected in the CSF and levels in the brain were relatively low [43], suggesting that the choroid plexus retains cadmium and limits its release to the CSF The most likely explanation is that MT binds the cadmium, a well-known property of this protein [4], although Zheng et al [43] did not detect MT in either the choroid plexus or the brain, likely because their assay system was of low sensitivity Others have shown that MT or MT-gene transcripts are present in the brain [7,8,10,1 l] and the choroid plexus ([39,44], present report)

J Hidalgo et al /Chem-Btol Interact 93 (1994) 197-219 215

4 3 Radtolmmunoassay o f MT- I zn spectfic bram areas From the above results, it is clear that Zn, Cu and glucocortlcolds can stimulate

MT-I accumulation in cultured neurons and astrocytes, suggesting that they play a role in the regulation of brain MT-I levels in VlVO There is already evidence for such a role for Zn [7-9], and we have now shown that 1 p rejection of dexamethasone affects MT-I accumulataon in 8 specific braan areas in the rat The distribution of MT-I in these areas in control rats was not homogeneous, which is m good agree- ment with previous reports [11,45] and wath the lmmunocytochemlcal results This may reflect functional differences that deserve further attention The response to dexamethasone was also non-uniform across these 8 brain areas since MT-I concen- trataons increased an some brain areas (cortex, cerebellum, pons + medulla, mid- brain and happocampus), but not m others (hypothalamus, frontal cortex and strmtum) It is of anterest to compare the effects of dexamethasone w~th those of stress on brain MT-I levels, since glucocortlcolds are inducers of the MT gene [2], are released during stress, and could therefore be medmtors of the MT-I response to stress Stress sigmficantly increases MT-I levels in frontal cortex, pons + medulla oblongata and hypothalamus, but not m mldbraln and h~ppocampus [11] Of these 3 areas sensitive to stress, only the p o n s + medulla oblongata responds to dex- amethasone Therefore, it seems unhkely that glucocortlcolds are general mediators of the bram MT-I response to stress, although they may have a role m other situations

Endotoxln is a potent inducer of MT synthesis in the hver and other t~ssues [46,47], a process hkely mediated by the release of cytokmes [47,48] which is rode- pendent of metals and glucocortacolds [49] Whereas it is clear that brain MT mRNA levels are increased by endotoxm [48], we have been unable to see an mcrease of MT- I levels in cortex and cerebellum 8 h after endotoxln admmlstrat~on In contrast, the same animals had the expected strongly increased hver MT levels, suggesting a dif- ferent response of both tissues The reasons for brain MT-I levels not being increased by endotoxln (since mRNA levels do mcrease) are unknown, but dlscrepanoes be- tween the concentrations of MT protein and mRNA concentrations have been noted in other tissues such as the endocrme pancreas [50] In agreement with the m vwo results, primary cultures of ghal cells &d not respond to endotoxln Furthermore, they &d not respond either to recombinant human IL-1 and IL-6 This suggests that cytoklnes have a hmited role m the regulation of brain MT concentrations

5. General conclusions

Several factors such as stress, metals (Zn, Cu) and glucocortlcolds have now been shown to increase brain MT levels in vwo and/or in vitro The functions of MT in these s~tuations are still unknown, but it seems likely that there will be more than one The nature of the metal(s) associated with MT in the different bram areas have not been estabhshed, but are most likely to be Zn and/or Cu, as is the case m periphencal tissues These metals are essential for normal bram function and it is Im- portant to keep the levels of free Zn and Cu in the cell within narrow limits Extracel- lular Zn levels can sometimes reach concentrations well above those stu&ed here,

216 J Htdalgo et al /Chem-Btol Interact 93 (1994) 197-219

e.g. 300 #M [51], and the present results may therefore be relevant to the in vIvo SltU- atmn. MT ~s a good candidate to chelate excess Zn and Cu and release them when needed for normal cell function. Furthermore, Zn-MT or Cu-MT could activate spe- cific enzymes, such as pyrldoxal kinase [52] and neurocupreln [53], respectively This does not mean, however, that parallelisms need exist between the concentratmns of Zn and/or Cu and MT levels m all brain areas [11] Comparison of the Zn levels of specific brain areas [54,55] and their MT content (this report), supports the view that higher Zn levels do not necessarily cause higher MT levels m basal conditions Other factors, such as glucocorticolds, may modulate the production of MT in the brain MT, on the other hand, could also exert an antmxldant function in some conditions [56] Further studies on the functions of MT in the brain are warranted

It has recently been reported that a growth inhibitory factor (GIF) in human brain has an amino actd sequence that is 70% identical to that of human MT-II [57] This has been confirmed later as a MT-III , an lsoform specific of the brain [58]. Unlike MT, GIF (or MT-III) was present in astrocytes but not in neurons and was present only in gray but not in white matter Moreover, rabbit MT, which is about 89% homologous to human MT, had no GIF activity at concentratmns well above those of GIF [57]. These findings imply that there are major &fferences between MT and GIF in their distribution and properties and it does not seem likely that our results could have been influenced by reaction between rat brain G I F and the anti-MT anti- bodies In support of this assumption, MT-I but not MT-II I m R N A levels were increased by dexamethasone [58] Furthermore, the antibodies used for MT-I measurements do not cross-react with human GIF, suggesting that the present re- sults are mostly reflecting MT-I levels Further studies about the factors controlling this brain specific MT isoform are warranted

Acknowledgements

The authors acknowledge the collaboratmn and generous gift of human GIF to Dr Y Uchlda, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, as well as the generous gift of recombinant human IL-I c~ and IL-6 to Hoffmann-La Roche and Genetics Institute, respectwely Thanks are given to Dr P. Kllle (University of Wales College of Cardiff) for the monoclonal E9 antibody. J H acknowledges the support of the Fondo de Investlgaclones Samtarias de la Segurldad Social (90/0065- 2-D) and that of the Direcc16n General de Investlgacl6n Clentifica y T&mca (PB91- 0489), and A G, that of the Dlreccl6n General de Investxgac16n Cientifica y T6cmca (PM89-0079) J H , M G , A G and I B acknowledge the support of the Accl6n Integrada Hlspano-Bntfinlca 055-A J H and H M acknowledge the support of the Commission of the European Commumtles [5893] A M O is a recipient of a predoctoral fellowship from Mlnlsteno de Educac16n y Clencla (Spare) Thanks are gwen to Mlguel Martll for his help in the lmmunocytochemlcal studies

References

1 R J Cousins, Absorption, transport and hepatic metabohsm of copper and zinc special reference to metallothlonem and ceruloplasmm, Physlol Rev, 65 (1985) 238-309

J Hidalgo et al/Chem-Biol Interact 93 (1994) 197-219 217

2 D H Hamer, Metallothionem, Annu Rev Biochem, 55 (1986) 913-951 3 I Bremner, Involvement ofmetallothlonem m the hepatic metabohsm of copper, J Nutr, 117 (1987)

19-29 4 J H R Kagl and Y Kojlma, (Eds), Metallothlonein II, Expenentm Suppl, Blrkhauser, Basel, 1987 5 R M Nalbandyan, Copper in brain, Neurochem Res, 8 (1983) 1211-1232 6 C J Fredenckson, G A Howell and E J Kasarkls, The Neuroblology of Zinc, part A, Alan R Llss,

New York, 1984 7 M Ebadl, Blochermcal characterization of a metallothioneln-hke protein in rat brain, Biol Trace

Elem Res, II (1986) 101-116 8 V K Pahwall, P L Iversen and M Ebadi, Regulation of zinc metallothioneln II mRNA level m rat

rain, Neurochem Int, 17 (1990) 441-447 9 M Ebadl and J C Wallwork, Zinc-binding proteins (hgands) in brains of severely zinc-deficient rats.

Blol Trace Elem Res, 7 (1985) 129-139 10 J Hidalgo, M Borras, J S Garvey and A Armarlo, Liver, brain and heart metallothloneln induc-

tion by stress, J Neurochem, 55 (1990) 651-654 II J Hidalgo, L Campmany, O Martl and A Armano, Metallothionein-I Induction by stress m speci-

fic brain areas, Neurochem Res, 16 (1991) 1145-1148 12 F Loffner, S M Lohmann, B Walckhoff, V Walter and H B Hamprecht, lmmunocytochemlcal

characterization of neuron-ennched primary cultures of embryomc rat brain cells by established neuronal and ghal markers and by monospecific antlsera against cyclic nucleotlde-dependent protein qumases and the synaptlc vesicle protein synapsin-I, Brain Res, 363 (1986) 205-221

13 L Agull6, F Plcatoste and A Garcia, Histamine sumulation of cychc AMP accumulation m astrocytes-ennched and neuronal primary cultures from rat brain, J Neurochem, 55 (1990) 1592-1598

14 J S Garvey and M F Heft, Separauon and collection of rat hver macrophages, Methods Enzymol. 108 (1984) 285-294

15 P O Seglen, Preparation of isolated rat hver cells, m D M Prescott (Ed), Methods in Cell Biology, Academic Press, New York, 1976, pp 29-83

16 D- L Knook, E C Sleyster and M J van Noord, Changes m lysosomes during ageing of paren- chymal and nonparenchymal liver cells In V J Chnstofalo and H Holeckova (Eds), Cell Impair- ment in Ageing and Development, Plenum Press, New York, 1975, pp 155-169

17 T Gasull, D V Rebollo, B Romero and J Hidalgo, Development of a competitive double antibody radiolmmunoassay for rat metallothionein, J Immunoassay 14 (1993) 209-225

18 R K Mehra and I Bremner, Development of a radlolmmunoassay for rat liver metallothioneln-I and its apphcatlon to the analysis of rat plasma and kidneys, Biochem J , 213 (1983) 459-465

19 M M Bradford, A rapid and sensitive method for the quantitatlon of microgram quantities of pro- tern utdlzlng the principle of protein-dye binding, Anal Blochem, 72 (1976) 248-254

20 B Jasam and M E EImes, Immunohlstochemical detection of metallothionein. Methods Enzymol, 205 (1991) 95-107

21 H G T Blaauwgeers, P A E Sillevis Smitt, J M B V de Jong and D Troost, Distribution of metallo- thlonein in the human central nervous system. Gha, 8 (1993) 62-70

22 J Hidalgo, A Dingman and J S Garvey, Role of extracellular zinc and copper on metallothionein regulauon in cultured rat hepatocytes, Hepatology, 14 (1991) 648-654

23 G R Magneson, J M Puvathmgal and W J , Jr Ray, The concentrations of free Mg 2+ and free Zn 2+ m equine blood plasma, J Biol Chem, 262 (1987) I ll40-11148

24 J Wensink, A J Molenaar, U D Woromecka and C J A Van den Hamer, Zinc uptake into synap- tosomes, J Neurochem, 50 (1988) 782-789

25 G A Howell, M G Welch and C J Fredenckson, Stimulation-induced uptake and release of zinc m hlppocampal slices, Nature, 308 (1984) 736-738

26 M Ebadl, T Takahashi and P Timmins, The stimulation of metallothlonein synthesis in neuroblastoma IMR-32 by zinc and cadmium but not dexamethasone. B~ol Trace Elem Res, 22 (1989) 233-246

27 P Thakran, M P Leuschen and M Ebadi, Metallothionein reduction in rat hlppocampal neurons In primary culture, In vivo, 3 (1989) 191-198

28 D E Hartter and A Barnea, Brain tissue accumulates 67copper by two hgand-dependent saturable

218 J Htdalgo et al / Chem-Btol Interact 93 (1994) 197-219

processes A high affinity, low capacity and a low affimty, high capacity process. J Blol Chem. 263 (1988) 799-805

29 B M Katz and A Barnea. The hgand specificity for uptake of complexed copper-67 by brain hypothalamlc tissue is a function of copper concentration and copper hgand molar ratio. J Blol C h e m . 265 (1990) 2017-2021

30 A Barnea. D E Hartter. G Cho. K R Bhasker. B M Katz and M D Edwards. Further characterization of the process of in vitro uptake of rad~olabeled copper by the rat brain. J Inorg Blochem. 40 (1990) 103-110

31 M L Fadla and R J Cousins. Zinc uptake by isolated rat liver parenchymal cells. Biochim Blophys Acta. 538 (1978) 435-444

32 M L Fallla and R J Cousins. Zinc accumulation and metabolism m primary cultures of rat l,ver cells regulation by glucocorticoids. Blochlm Blophys Acta. 543 (1978) 292-304

33 A L Wemer and R J Cousins. Copper-accumulation and metabolism m primary monolayer cul- tures of rat liver parenchymal cells. Blochim Blophys Acta. 629 (1980) 113-125

34 L W Swanson. D M Simmons. J Amza . R Hammer. R Brmster. M G Rosenfeld and R M Evans. Novel developmental specificity m the nervous system of transgemc animals expressing growth hormone fusion genes. Nature. 317 (1985) 363-366

35 N Nishlmura. H Nlshimura and C Tohyama. Metallothionem local,zatlon m cadmium-treated rat brain. J Histochem Cytochem. 38 (1990) 1023

36 J K Young. J S Garvey and P C Huang. Ghal lmmunoreactwity m the rat brain. Gha. 4 (1991) 602-610

37 N Nlshlmura. H Ntshimura. A Ghaffar and C Tohyama. Localization of metalloth~oneln m the brain of rat and mouse. J Histochem Cytochem. 40 (1992) 309-315

38 K Suzuki. K Nakajima. U Kawaharada. F Hara. K Uehara. N Otakl. M Klmura and Y Tamura. Localization of metallothlonem in the brain of Maca~a fas(t¢ularts. Acta H,stochem Cytochem. 25 (1992) 609-616

39 K Suzuki. K Nakajlma. U Kawaharada. K Uehara. F Hara. N Otak~. M Klmura and Y Tamura. Metallothlonem m the human brain. Acta Hlstochem Cytochem. 25 (1992) 617-622

40 K Nakallma. K Suzuki. N Otakl and M Klmura. Detection of metallothloneln in brain. Methods Enzymol. 205 (1991) 387-395

41 M Panemangalore. D Banerjee. S Onosaka. M G Chenan. Changes m the lntracellular accumula- tion and distribution of metallothlonem in rat liver and kidney during postnatal development. Dev Blol. 97 (1983) 95-102

42 N Nlshlmura. H Nlsh~mura and C Tohyama. Immunoh~stochem~cal locahzat~on of metallothlon- em In developing rat tissues. J Hlstochem Cytochem. 37 (1989) 715-722

43 W Zheng. D F Perry. D L Nelson and H V Aposhian. Choroid plexus protects cerebrospmal fluid against toxic metals. Faseb J . 5 (1991) 2188-2193

44 Y ltano. S Nojl. E Koyama. S Tamguchl. N Taga. T Takahashl. K Ono and F Kosaka. Bacter- Ial endotoxm-mduced expression of metallothlonem genes in rat brain, as revealed by in sltu hybrid- ization. Neurosci Let t . 124 (1991) 13-16

45 S M Sato. J M Frazier and A M Goldberg. The distribution and binding of zinc m the h,ppocam- pus. J Neurosci . 4 (1984) 1662-1670

46 I Bremner. J M M o m s o n . A M Wood and J R Arthur. Effects of changes m dietary zinc. copper and selemum and of endotoxln administration on metalloth~onem 1 concentrations in blood cells and urine m the rat. J Nu t r . 117 (1987) 1595-1602

47 J Llu. Y P Lm. L E Sendelbach and C D Klaassen. Endotoxm mduct~on of hepatic metalloth~on- em is mediated through cytokmes. Toxlcol Appl Pharmacol . 109 (1991) 235-240

48 S K De. M T McMaster and G K Andrews. Endotoxm reduction of murme metallothloneln gene expression. J Blol C h e m . 265 (1990) 15267-15274

49 D M Durnam. J S Hoffman. C J Quaife. E P Bendltt. H Y Chen. R L Brmster and R D Palmiter. Induction of mouse metallothlonem-I m R N A by bacterml endotoxln is independent of metals and glucocortico~d hormones. Proc Natl Acad Sc~ USA. 81 (1984) 1053-1056

50 G K Andrews. K Kage. P Palmiter-Thomas and M P Sarras. Metal ions reduce expression of metallothlonem in pancreatic exocrme and endocrine cells. Pancreas. 5 (1990) 548-554

J HMalgo et al /Chem-Btol Interact 93 (1994) 197-219 219

51 S Y Assaf and S -H Chung, Release of endogenous Zn 2+ from brain tissue during actwlty, Nature, 308 (1984) 734-736

52 J E Churchlch, G Scholz and F Kwok, Actwatlon of pyndoxal kmase by metallothlonem, Blochlm Biophys Acta, 996 (1989) 181-186

53 V Z Melkonyan, M V M~kaelyan and R M Nalbandyan, Reconstltutlon of neurocuprems by Cu- thlonems, Neurochem R es , 14 (1989) 589-591

54 P Szerdahelyl and P K~isa, Partial depletion and altered distribution of synaptlc zinc m the rat hlp- pocampus after treatment with sodium dlethyldlthlocarbamate, Brain Res , 422 (1987) 287-294

55 K Gulya, G L Kov;ics and P Kasa, Partial depletion of endogenous zinc levels by (D-Pen 2, D- Pen 5) Enkephahn in the rat brain, Life Sc~, 48 (1991) PL57-PL62

56 J Hidalgo, L Campmany, M Borras, J S Garvey and A Armano, Metallothlonem response to stress m rats role m free radical scavenging, Am J Physlol , 255 (1988) E518-E524

57 Y Uchlda, K Taklo, K Titam, Y Ihara and M Tomonaga, The growth inhibitory factor that is deficient m the Alzhelmer's d~sease brain ~s a 68 ammo acid metalloth~one,n-hke protein, Neuron, 7 (1991) 337-347

58 R D Palmlter, S D Fmdley, T E Whltmore and D M Durnam, MT-III, a brain-specific member of the metallothlonem gene family, Proc Natl Acad Scl USA, 89 (1992)6333-6337