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Neurohehaxioral

Toxicology and

Tera'ioli 

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iona l Inc . Prime d i n Ih c U.S.A . 0:75-l?W)/85$J.O O + .0 0 

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# Neurobehaviora l an dNeurochemica l Abnormalitie s o f

Pre

- an dPostnatall y Lead-Expose dRats : Zinc , Coppe r an d Calciu mStatu s

MARI O BARALDI, 1 

### PAOL A ZANOLI , TIZIAN A ROSSI , PAOL A BORELLA, *

EV A CASELGRANDI * AN D FELIC E PETRAGLIA t Institute of

Pharmacology , Chair

of Hygiene,* Department

of Obstetrics and

Gynecology^ University of

Modena, 41100 Modena, Italy

BARALDI , M. , P. ZANOLI , T . ROSSI . P . BORELLA , E . CASELGRANDI AN D F . PETRAGLIA . Neurobehavioral and neurochemical

abnormalities of

pre- and

postnatalh lead-exposed rats: Zinc, copperand calcium status. NEUROBEHA V TOXICO L TERATOL 7(5)499-509 , 1985.—Althoug h divergin g opinion s hav

e bee n expressed , ther

e i s a bod y o f evidenc e o f

a cause-effectrelationship between chronic exposure to lea d an d altere d behavio r i

n children . Ther

e ar e several problems i

n the interpretatio

n o f the behaviora

l an d biochemica

l anomalies described i

n experimenta

l model s o f lea d intoxicatio n du

e mainl y t o the.differen

texperimenta

l condition s used . On

e o f the majo r criticism s i s linke

d wit h th e clai m tha ti n severa l studie s to o hig

h level s o f exposure wer

e used , henc

e inducin g the suspicio n tha

tth e effects specifi cally attribute

d to lea d intoxicatio n coul d b e relate d to unspecifi c actions . Her e we repor

tthat, usin

g a n experimenta

l mode l o f pre - an d postnata

l intoxicatio n o f

rat s expose d to a ver

y low amoun

to f lea d (I mg/kg/day) , i

n paralle l wit h behaviora

l anomalies , ther

e ar e slightchange s i

n cerebra l dopaminergi

c an d GABAergic receptors . A ne w findin g was the discovery tha ti n thi s conditio n ther

e i s a markedl

y increased numbe

r o f opiat e receptors i

n the hypothalamu

s (an d other brai

n areas ) i n paralle l wit h a decreas e o f

/3-endorphi

n an d a n increas e o

f Met-enkephalin . Therefore , low level s o f lea d intoxicatio n whic h coul d b e pertinen

tto the huma

n situation , seem to induc e neurotoxi c effects whic h ma y b e responsibl e fo

r th e behaviora l anomalie s i

n rat s and , maybe , i

n children . Analgesi a Dopamin

e receptor

s /3-Endorphi

n GABA receptors Met-enkephali n Lea

d nitrat e Locomoto r activit

y Morphin

e Opiat

e receptors Perinata

l exposure Sodiu

m nitrat e EXPOSUR E t o hig h level s o f lea d induce s a n encephalopathi c stat e characterize d b y brai n hemorrhages , seizure s an d com a (fo r a revie w se e [34]) . Ther e ar e contro versia l report s o n th e pathogeneti c rol e o f lea d i n s o calle d "minima l brai n dysfunction " (MBD) , a syndrom e whic h mainl y affect s childre n an d i s characterize d b y hyperactiv ity , shor tattentio n spa n an d eas y distractabilit y [22-24] . I n < pas tattempt s t o reproduc e th e syndrom e experimentall y ther e wa s littl e concer n t o us e dose s o f lea d whic h coul d b e relate d t o th e huma n situation , wher e hyperactivit y become s apparen tafte r asymptomati c lea d intoxication . A tth e sam e tim e i ti s ver y difficul tt o compar e th e behaviora l an d biochemica l effect s o f

lea d intoxicatio n reporte d i n differen t paper s becaus e o f

th e grea tvariabilit y i n th e dos e o f lead , rout e o f administration , duratio n o f exposure" , etc . Critica l point s see m t o b e relate d t o th e us e o f hig h dose s o f

lead , whic h ma y lea d t o nonspecifi c effects , a s wel l a s lac k o f concer n fo r secon d io n effects , th e dat e a twhic h exposur e t o th e meta l began , th e ag e a twhic h behaviora l an d biochemi ca l test s wer e performed , variabilit y du e t o sex , monitorin g o f lea d level s i n bloo d an d brai n tissues , interactio n wit h othe r cation s and , las tbu tno tleast , accurat e assa y oflea d i n biologica l specimens . Le tu s briefl y discus s a fe w o f thes e points . A s fa r a s th e leve l o f chroni c lea d exposur e i s concerned , i tmus tb e pointe d ou ttha ti n man y studie s o f "lo w level " lea d toxicit y th e dose s oflea d use d ar e s o hig h (10-20 0 mg/kg/day ) tha tth e relevanc e o f

th e result s t o subclinica l lea d poisonin g i n chil dre n i s questionable . I tha s bee n argue d i n fac ttha tth e ob serve d behaviora l an d biochemica l alteration s i n som e o f thes e experiment s coul d i n fac tresul tfro m malnutritio n [17,53 ] an d inhibitio n o f energ y metabolis m [16] . O n th e basi s o f

clinica l studie s i n childre n [42,48 ] 30 0 ju. g o f elementa l lea d i s regarde d a s th e maximu m dail y permis sibl e intak e fro m al l source s an d doublin g o f thi s dos e i s enoug h t o produc e advers e metaboli c effects . Bloo d level s rangin g betwee n 1 0 an d 2 0 /xg/d l hav e bee n describe d a s inducin g a n alteratio n o f hem e synthesi s [18,66 ] i n lead expose d children . In rat s bloo d level s o f 1 1 jug/d l hav e bee n describe d a s resultin g i n detectabl e histologica l lesion s i n 'Request s fo r reprint s shoul d b e addressed t o Prof . Mari o Baraldi , Associate Professo r o f Toxicology , Institut

e o f Pharmacology , Univer sit y o f Modena , Vi

a Camp i 287 , 4110

0 Modena, Italy .

V ' 49 9P. 3

50 0 

### BARALD I ETAL. TABL E 1 EXPERIMENTAL DESIG N O F CHRONI

O L.EA D EXPOSUR

E O F RAT DAM

S DURING GESTATIO

N A N D LACTATIO N • •-- - •- , Dams mate d 1 2 1 2 1 2 Pregnan

t 6/1 2 5/1 2 8/1 2 Treatmen

t wate r NaNO , Pb(N0 3 ) s ) Dos e

mg/k g daily



### — 1 1 Volume ml/k g 2 2 2 Dosin g
ora l
ora l
ora l System gastri c intubation gastri c intubation gastric intubation Duratio

n o f treatmen t Week s 6 6 6



### rena l proxima l tubul e cell s [28] . I

n immatur

e rat s greater lead toxicity tha

n i n adult

s ma y b e cause d b y higher retentio n [43] , reduced eliminatio n rat e [39 ] an d preferentia

l localiza tio n i n cerebra l tissue s [55] . Moreover , itha s been reporte

d tha tth e immatur

e CN S i s particularl

y vulnerabl e t

o toxi c effects o f lea d [34] . Fro

m thes e dat

a on e ca n surmis e tha

t ther e i s n o reaso n t

o expos e animals t

o larg e amount s oflea d i n orde r t o mimi c th e toxi c effects (MBD ) induced i n children b y pre - an d postnata

l exposur e t o asymptomati c doses . I n th e mos twidel y use d anima l mode l o f chroni c lead poisoning , exposure i s achieve d vi a th e drinkin g wate r by usin g lead acetate [12,13] . Howeve r the us e oflea d acetate coul d b e questione d sinc e th e aceti c aci d componento f lea d acetate ha

s been shown t o b e capabl e o f maskin g th e effects oflea d o n bot

h bod y weightan

d locomoto r activit

y [9] . Fro m thi s las tfindin

g i ti s clearl y imperative t o contro l th e effects o f the secon d io n o f

th e lea d compound s used , espe ciall y whe n hig

h dose s oflea d salts ar

e administered . Severa

l anima l studie s hav e bee n carrie d outo n th e effecto f

lead acetate o n th e activit

y o f ra tpup s expose d pre - an d postnatally , bu

tsom e authors reporte

d no change i

n leve l activit y [44,64] , others hypoactivit y [25,65 ] an

d others hyperactivit

y [30,57 ] attributable t o lead . If methodologica

l difference s among experiment s ma y contribut

e t o thes e in consistencies , i tcoul d wel l b e tha tth e aceti c componen to f lead acetate playe d a par tin th e variabilit

y o f th e results . Various anima

l experimenta

l model s hav e bee n devel ope d t o investigate th e neurobehaviora l an d neurochemica

l effects due t o lead exposur e durin g prenata l an d postnata

l life . Thes e anima

l model s o f

lead intoxicatio n have produced a ver y complicate d literature , a s recentl y pointed outb y Winde r an d Kitchen [82] , wh o reviewe d th e biochemica l effect s whic

h have bee n describe d a s bein g associate d wit h lead toxicit

y in th e CNS . Three majo

r system s o f

neurotransmissio n hav e bee n ex tensivel y studied : the dopaminergic , th e cholinergi c an d th e GABAergi c system , whil e les s attentio n ha s bee n devote d t o th e brai n opiatergi c system . Several studie s have provided evidenc e o f

a catecholaminergi c dysfunctio n due t o lead poisoning , bu

tther e ar e conflictin g results sinc e bot h inhibi tio n an d stimulation o f

dopaminergi c functio n have bee n re ported . Thes e discrepancie s have bee n attributed t o difference s in duration o f

exposur e an

d dose s o f

lead adminis tered , suggestin g tha

thig h dose s o f

lead inhibi

tth e dopaminergic syste m whil e relativel y lo w dose s activate i t [81,82] . Thoug

h thi s hypothesi s need s furthe r experimenta

l demonstratio n iti s noteworthy i n thi s contex ttha

tth e oppo site regulation o f dopamine level s i

n differen tbrai n areas reported by Govoni et

al. [31,32 ] i

n pre - an d postnatally lead expose d rat

s (2. 5 g/ 1 i n drinkin g water) coul d well ex plain the variable results reported i n th e literatur

e o n dopamine turnover . A s fa r as dopaminergi c recepto r studie s ar e concerned , Lucchi et

al. [45 ] have describe d a n in crease d densit

y o f receptors i

n th e striatu

m (where dopamine synthesi s wa s reduced ) an d a down-regulatio n i

n th e nucleu s accumben s (where dopamine synthesi s was increased ) whe n 3

### H-sulpirid e ( a ligan

d believe d t

o b e specifi c for dopamine D , receptors ) was used , whil e n o chang e wa s foun d usin g 'Hspiperon e ( a ligan

d whic h bind s bot

h D , an d D , receptors bu

t wit h preferentia

l labeling o f

D 2 

### ) . Th e chroni c decrease d an d enhance d presenc e o f

dopamine i

n th e tw o nuclei would b e responsibl e for the increase d an d decrease d numbe

r o f dopamine receptors i

n th e tw o area s describe d respectively . Severa l studie s (for a revie w se e [71,82] ) hav e been performe d t o demonstrat

e th e possibl e implicatio n o f functiona l change s i n th e cholinergi c syste m i

n lea d toxicity . However , sinc e the hypofunctio n o f thi s syste m reportedly wa s linked onl y t o exposur e t o ver y hig h dose s o f

lead , itha

s bee n suggeste d tha

ti ti s unlikely tha

tlead has a specifi c neuro chemical toxi c effecto n thi s syste m [82] . Studie s o n brai n GAB A metabolism i

n anima l model s usin g ver

y hig h dose s o f lea d ( 5 g o r 1

0 g Pb/ I i n drinkin g water , equivalen tt

o abou t 118-12 6 mg/kg

) have describe d reduce d uptak e an d releas e o f GABA , change s i

n GABAtransaminas e an d glutami c aci d decarboxylas e a s well a s increase d GAB A synthesi s [72] . Th e effects oflea d o n GABAergi c enzymes , however , were notconfirme

d by Govoni et

al. [31 ] usin g a lower , bu tstill hig h dos e oflea d (2. 5 g/1 , equivalen tt o about5 4 mg/kg/day) . I n th e sam e study Govoni et al. [32 ] reported a

n increas e in GAB A receptors i

n th e cerebellu m an

d a decreas e i

n th e striatum . I

tha s been suggeste d tha

tth e neurotoxicit

y o f lea d coul d b e partiall

y related t o th e lead-induce d increas e in delta-amino-levulini c acid , which i s a wea k displace r o f GAB A bindin

g [74] . I ti s tempting t o sugges ti n thi s regar

d tha ta n increase d chroni c presenc e o f thi s agen tcould , di rectl y o r indirectly by stimulatin g GAB A releas e an d inhibiting GAB A uptak

e [14,15] , down regulat

e GAB A receptors i

n certai n brai

n areas . Finally , th e effecto f

lead intoxicatio n o n th e opiatergi c syste m has onl y been studie d in th e striatu m b y measuring Met-enkephalin levels . While Govoni et al. [32 ] and Mem

o et al. [51 ] hav e reporte

d a n increas e in thi s neuropeptid e after 9 0 day s o f

lea d acetate exposur e (2. 5 g/1 , equivalen tto 135 0 pp m o r abou

t5 4 mg/kg/day) , Clayto n et at. [19 ] foun

d de -P. 4

LOW-LEVE L LEA D NEUROTOXICIT Y Mi l TABL E 2 EFFEC T O F LEA D EXPOSUR

E O N GESTATIO

N AN D PARTURITIO

N % o f Bod

y weigh t Treatmen tDay s o

f Numbe

r o f die d or (g

) o f pup s ( I mg/kg/os/day) gestatio n pups malforme d pups a

tbirt h water NaNO , PrXNO,)., 21.8 1 ±0.1 8

22.6 0 ± 0.2 4 22.1 3 ± 0.3 0 12.67 ±0.6 7

8.20 ± 2.0 6 10.38 ± 0.9 1 6.1 4 ± 0.1 1 6.55 ± 0.32 6.2 3 ± 0.44 Value s ar e reporte d a s mea n ± S.E.M .
TABL E 3 LEAD , ZIN C AN D COOPE RCONCENTRATION

S I N URINE S O F LACTATIN

G MOTHER S POISONE

D WIT H LEA D ( I mg/kg/day/os

) DURIN G PREGNANC

Y AN D LACTATIO

N Concentrations i

n urines § (mea

n ± SEM )
Treatmen t Zin

c (Mg ) Coppe

r (Mg ) Lea

d (ng ) Urin

e output/ra t (ml ) Mea n ± S D
wate r NaNO , PlHNO^ j 2.1 9 ±0. 4 2.6 4 ± 0. 5 3.1 8 ±0. 2 1.5 1 ± 0. 3

1.1 1 ±0.0 9 1.5 5 ± 0. 3 13. 0 ± 3. 5

9. 3 ± 0. 3 83. 3 ±14.5 * 5. 6 ± 1. 2 4. 0 ± 1. 8 5. 3 ± 1. 3 SUrin e wa s collecte d for 6 h

r fro m rat s i n metaboli c cages . Student' s r-Test : *p<0.001 . 

# I

r ~ crease d enkephali n level s afte r exposur e t o lowe r dose s o f

lea d (30 0 an d 10 0 ppm ) an d fo r a shorte r perio d (fro m con ceptio n t o weaning) . I n thi s regar d i ti s als o interestin g t o mentio n tha taltere d response s t o morphin e hav e bee n re porte d i n rat s b y Mitchel l et al. [54 ] afte r a n acut e treatmen t wit h lead . Al l thes e importan tobservation s take n togethe r see m t o indicat e tha ta n imbalanc e betwee n differen texcita tor y an d inhibitor y neurotransmissio n system s coul d b e operativ e i n th e neurobehaviora l effect s oflea d intoxication . I n attemptin g t o explai n th e multiplicit y o f effect s induce d b y lea d o n th e CNS , littl e concer n ha s bee n devote d t o th e possibilit y tha tsom e o f th e neurotoxi c effect s o f lea d migh t b e du e t o interferenc e wit h homeostasi s o f othe r divalen t cation s suc h a s calciu m an d zinc . I tha s bee n suggeste d tha t th e effect s o f

lea d o n monoaminergi c function s coul d b e re late d a tth e neurona l leve l t o inhibitio n o f mitochondria l cal ciu m uptake : whethe r thi s effec tresult s i n a n increase d o r decrease d calcium-dependen treleas e o f dopamin e i s stil l unde r debate , sinc e experimenta l suppor tfo r bot h thes e op posit e effect s ha s bee n provide d i n differen tmodel s o f lea d intoxicatio n [73 , 80 , 81] . Further , exposur e t o dose s oflea d rangin g fro m 20 0 t o 135 0 pp m [46 , 47 , 52 ] ha s bee n describe d a s depressin g tissu e concentration s o f zin c an d a s increasin g urinar y zin c excretio n [79] , a n effec twhic h coul d b e linke d wit h th e wel l prove d rena l toxicit y exerte d b y thes e dose s o f lead . AI M Th e purpose s o f

ou r stud y were : (1 ) t o fin d a threshol d dos e o f lea d whic h coul d mimi c i n pre - an d postnatall y ex pose d rat s th e hyperactivit y syndrom e describe d i n children ; (2 ) t o perfor m atth e sam e tim e neurobehaviora l an d neurochemica l assessmen ti n th e brai n b y studyin g th e func tiona l statu s o f

neurotransmitte r an d neuropeptid e systems ; (3 ) t o monito r th e level s o f lea d i n urine , bloo d an d brai n area s togethe r wit h th e level s o f othe r divalen tcation s suc h a s zinc , coppe r an d calcium , whic h see m t o modulat e recep to r function s i n th e brai n [6 , 21 , 50 , 59 , 75 , 77] .

ANIMA L MODEL

S I n vie w o f

al l th e abov e mentione d consideration s an d dat a fro m ou r preliminar y studie s o n lea d accumulatio n i n . th e bloo d an d brai n afte r exposur e t o varyin g dose s oflead , w e decide d t o stud y th e effect s o f a ver y lo w dos e o f

lea d o n ra tpup s pre - an d postnatall y expose d throug h materna l dos ing . Fo r thi s purpos e femal e Wista r rat s wer e treate d dail y b y gastri c intubatio n wit h 1 mg/k g o f P b (N0 3 ) 2 fro m matin g t o th e en d o f

lactatio n (Tabl e 1) . Th e offspring , afte r wean ing , underwen tth e sam e treatmen ttil l 6 0 day s o f age . Tw o othe r group s o f

rat s use d a s control s wer e treate d wit h NaNO. , t o tes tfo r th e secon d io n effec to f th e P b (N0 3 ) 2 an d wit h deaionize d wate r t o tes tfo r th e effec to f th e solven t alone . RESULT S AN D DISCUSSIO

N Th e leve l oflea d exposur e use d i n thi s stud y di d no taffec t th e bod y weigh to f

th e dam s durin g gestatio n an d lactation , th e duratio n o f

pregnanc y o r th e numbe r o r weigh to f litter s delivere d i n compariso n wit h control s (Tabl e 2) . A s reporte d elsewher e [8] , n o significan tdifference s wer e

### 50

2 BARALD I ET AL.

see n betwee n lea

d expose d dam

s an d control s a

tthe en d o f the nursin g period i n urin e an d plasm a concentration s o f

zin c an d coppe r determined by a n atomi c absorptio n spec trophotometri c method previousl y describe d [5 ] (Table s 3 an d 4) , whil e th e excretio n oflea d was increase d abou t8 fold (Tabl e 3 ) an

d leve l o f lead in whol e bloo d was practically double d (Table 4) . Lead was measure

d b y a n atomi c absorptio n spectrome te r (Perkin-Elmer me

d 5000) equipped wit

h HG A 40 0 graphit e furnace , followin g th e procedur e o f Fernandez [27] . Th e reliabilit

y o f our metho

d wa s controlle d [78 ] by the Healt h an d Safet y directorat

e o f th e Communit

y Europea

n Commissio n (C.E.C.) . Thes e dat a ar e o f valu e i n order t o ensure tha

ta tthi s leve l o f exposur e lead doe s notinduc e an y apparen

tsignifican

t change in adul

tpregnan tfemal e rats . N o significan tdiffer ence s wer

e noted i n growt h (Table 5) , developmen to f th e righting refle

x o r ag e o f

ey e opening (dat

a notshown ) be twee n lead expose d an

d contro l pup s durin

g th e nursin

g period . I n thi s contex titi s noteworthy tha tonl y a few day s afte

r weanin g (3 0 day s o f

age ) th e pup s directly expose d t o lead by intubation showe d a statisticall y significan tdecreas e in body weigh twhen compared wit h controls . Sinc e thi s difference wa s no longe r apparen

ta t6 0 day s o f

age (Table 5) , i

ti s likel y tha tthi s phenomenon i s t

o b e attribute

d t o a greater reactio n tha n control s oflea d expose d pup s t o th e weaning-associate d stres s an d t o manipulations linked wit h th e gastri c intubation procedure o f

th e treatment. A n open-fiel d tes tperforme d a t1 5 day s o f age (Tabl e 6 ) showe d a significan

tincreas e i

n bot h numbe r o f crossed square s an

d pivot s in th e lead exposur e group i n comparison wit h bot h group s o f

controls . Th e open-fiel d tes twa s re peate d a t4 0 day s o f

ag e an d showe d practically th e sam e results , bot

h mal e an d female lead-treated rat s being mor

e activ e tha n controls . Howeve r comparison betwee n group s showe d a significan

tdifferenc

e onl y in th e grou

p o f male rats . Thes e dat

a see m t o b e simila

r t o huma

n response s t o lead , sinc e in th e huma

n situation , boy s exhibi thyperkineti c syndrome mor

e frequentl

y tha n girls [83] . Th e hyperactivit y o f lead-treated rat

s a t4 0 day s o f

age was confirme

d b y measuring locomoto r activit y in photocell-equippe d boxe s durin g 5 minut e test s repeated 3 time s a

tinterval s o f

3 0 min ute s an d evaluatin g onl y th e dat a o f th e las ttes tin orde r t o avoi d adaptation an

d curiosit

y interference s wit

h the locomotio n paramete

r (dat a notshown) . Thes e finding s take n togethe r see m t o demonstrat e tha tdaily exposure t o 1 mg/kg/da y o f lead nitrat

e durin g pre - an d postnata

l lif e in duce s border-line behaviora l effects whic

h d o notappea

r t o b e related t o nonspecifi c effect s du e t o genera

l toxicity or inhibition o f

energy metabolism , bot

h i n the nursin

g dam s an d in th e offspring . A s show n i

n Table s 8 an

d 9 urinar y excretio n oflea d at4 0 day s o f ag e wa s increase d abou

t7 fol d i n lead-expose d pups , whil e th e whol e bloo d leve l wa s twic e a s hig

h a s i n controls . Whe n compared wit

h the leve l observe d i

n the dams , thes e dat a indicat e a n equivalenc e between th e materna l dosin g an d th e direc

ttreatmen

to f the pup s wit

h lead . Further, th e level s oflea d in 4 brai

n areas teste d (Fig . 1) were foun d t o b e about8 time s highe

r tha n i n controls , a finding whic h con firm s th e tendenc

y o f lead [55 ] t

o accumulat

e i n the brai n durin g the early stage s o f

pre - an d postnata

l lif e alread y de scribed . O

n th e othe r han

d n o statisticall y significan t change s i n urinar y excretio n o f

coppe r and zin c were foun d in th e lead-expose d pups , a finding whic h seem s t o indicate , TABL E 4 CONCENTRATIONS OF ZIN C AN D COPPE RI N PLASM

A AN D OF LEA D I N WHOL E BLOO D O F LEAD-POISONE

D MOTHE

RRAT S DURING GESTATIO

N AND LACTATIO

N ( I mg/kg/DA

Y B Y ORA L INTUBATION)

Treatmen t Wate r NaNO , Pb(N0 3 ) , Value s ar

e *p<0.05 . Plasm a (Mg/10 0 ml ) Zin c Copper 133. 0 ± 1. 8 128. 6 ± 4. 4 119.7 ± 5. 3 reporte d a s 117. 6 ± 2. 9

116. 0 ± 5. 1 125. 7 ± 5. 5
mea n ± S.E.M. ; Whol

e Blood (Mg/10 0 ml ) Lea d 2.63 * 0.33 2.7 0 ± 0.3 6 5.8 7 ± 0.96 * Student' s r-Test :



### alon g wit h th e norma

l urin e output , tha

tthi s leve l o f

lea d exposur e doe s notresulti n overtrena

l toxicity . While no change was foun d i n plasma coppe r concentra tions , th e leve l o f

plasma zin c was foun

d t o b e enhanced i

n th e lead-expose d group . Thi s increase d presenc e o f circulating zin c was quit

e surprisin

g bu twas corroborated b

y the finding tha

tzin c wa s increased als o i n th e 4 brai

n area s teste d (Fig . 1) . A modes tincreas e o f zinc , whic

h di d not reac h statistica

l significance , wa s foun

d als o i n the cerebel lum , hippocampus an

d cortex o f

NaN0 3 

### treated rats . Moreover , a

n increase d presenc e o f

calcium , whic

h was statisticall y significan

ti n cerebellu

m an d cortex , was foun

d a tth e same age (4 0 days ) i n th e brain

s o f lead-expose d rats . Sinc e n o change i

n the leve l o f

coppe r wa s foun

d i n th e bloo d o r in th e 4 brai

n area s tested , i ti s likel y tha

tth e increased zin c an d calciu m ar e no tdue t o a lead-induce d genera

l in creas e i n divalentcation s enterin g acros s th e gu tan d blood brai n barrier, bu

trathe r t o specifi c mechanism s tha tneed furthe r elucidation . Th e us e o f

highe r dose s oflea d acetate ha s been describe d a s inducing n o change o r indee d a decreas e i n zin c homeo stasi s in th e brain : itcoul d b e that , a s suggeste d fo r othe r lead-induced phenomena [82] , a ver y lo w leve l oflea d expo sur e ma y produc e opposite effects t

o thos e foun

d after expo sur e t o hig h levels . I n thi s regar

d i ti s noteworthy , however , tha tthe contento f

zin c an d calcium , teste d i

n the whol e brai n o f 6 0 da y ol d lead-expose d rats , appeared t o b e norma

l in comparison wit

h th e value s o f

contro l rat

s (Tabl e 10) . Whateve r th e mechanis

m b y whic h exposure t

o lo w dose s o f lea d result s i n increase d presenc e o f

zin c an d calcium , i

ti s clea r fro m th e las tfinding mentione d tha tthi s lead-linked phenomenon is confined t o th e early stage s o f

pre - an d postnata l life . Th e findin

g tha ta lo w leve l o f

lead exposur e was able t o induce threshold behaviora l effects withoutimplication o f apparen tmalnutritio

n problems prompte

d u s t o stud y the functiona l activit y o f som e o f th e neurotransmitter system s previousl y suggeste d a s being implicated i n th e neurotoxicit

y o f lead . A s regard s th e functiona

l activit y o f brai n GABAA 

### recep tors , whic

h wer e studie d accordin g t o a method previously described using 'H-GABA as a ligan d [2] , w e foun d (Table 11) tha texposure t o th e lo w leve l o f lead w e used signifi cantl y reduced onl y th e lo w affinit y componento f

th e sodium-independen

t'H-GAB A bindin

g saturation curve s performe d i n cortica l brai n membranes . Thi s finding allowe d A IP. 6



### LOW-LEVEL LEA D NEUROTOXICIT Y 50 3



### TABL E 5 BOD Y WEIGH

T OF PUP

S NOURISHE

D BY LEAD-POISONE

D MOTHER

S Mothe r dail y treatmen t ( 1 mg/kg/os ) Wate r NaNO , Pb(NOa) 2 4 m 11. 7 ± 0. 6 11. 4 ± 0. 5 10. 8 ± 0. 5 Bod y
8 m 17. 4 ± 0. 5 18. 0 ± 0. 3 16. 7 ± 0. 3 weigh t(g ) o f pup s

32. 4 ± 0. 4 31. 8 ± 0. 6 30. 0 ± 0. 6 a tday :

24 , h
51. 7 ± 1. 1 51. 7 ± 1. 4 49. 6 ± 1. 3 30 , h

68. 2 ± 2. 1 64. 9 ± 1. 8 58. 7 ± 1.3 * 6

232. 4 ± 7. 2 235. 0 ± 8. 5 238. 6 ± 8. 8 60 , h 9

165. 3 ± 9. 4 178. 3 ± 12. 0 175. 7 ± 6. 6 Value s ar e reporte d a s mea n ± S.E.M. : Student' s '-Test : *p<0.0 1 vs . controls .


### TABL E 6 OPE N FIELD BEHAVIO

RO F BABY RATS (1 5 DAY S OF AGE ) NOURISHED B Y LEAD-POISONE D DAMS Treatmen tTes tses - Pivot s ( 1 mg/kg/o s No . o f siontim e Entrie s (mea n ± daily ) teste d pup s (min ) (mea n ± S.E.M. ) S.E.M. )
wate r NaNO , Pb(NO,) 2 18. 3 ± 5. 5 22. 4 ± 3. 5 30. 3 ± 4.1 * 2. 2 ± 0. 3

3. 4 ± 0. 6 4. 0 ± 0.5 * Student' s /-Test : *p<0.0 1 vs . controls .



### TABL E 7 OPE N HEL D BEHAVIORO F 4 0 DAY S OLD FEMAL E AN D MAL

E RATS NOURISHE

D BY LEAD-POISONE

D MOTHER

S AN D TREATE D FO R1 0 DAY S WIT

H LEA D B

Y ORA L INTUBATIO

N Treatmen t ( 1 mg/kg/o s daily )
wate r NaNO , PtKNO,) , No . o f

teste d pup s000000 Tes t

sessio n tim e (min ) 3 3 3 Mal e

Entrie s Pivot s 12. 7 ± 6. 3 2. 5 ± 0. 2 14. 7 ± 7. 1 3. 2 ± 3. 7 28. 5 ± 7.4 * 4. 5 ± 1.9 * Femal e Entrie s Pivot s 36. 2 ± 9. 7 36. 2 ± 5. 9 43. 5 ± 10. 1 6. 5 ± 1. 2

5. 4 ± 1. 8 8. 2 ± 2. 5 Al l reporte d value s ar e mea n ± S.E.M . Student' s /-Test : *p<0.0 1 vs . controls . 

### 5 *

= • % 

### TABL E 8 LEAD , ZIN C AN D COPPE RCONCENTRATION

S I N URINE S O

F PUP S (AGE D 4 0 DAYS ) NOURISHED BY LEAD-POISONE D MOTHER

S TILL WEANIN

G AN D EXPOSE

D FO R1 0 DAY S T

O LEAD ( 1 mg/kg/DA

Y B Y ORA L INTUBATION)

Treatmen t Concentrator ! i n urines § (mea n ± SEM )

Zin c (Mg ) Coppe r

(Mg ) Lea d

(ng ) Urin e

output/ra t (ml ) Mea n ± S D
wate r NaNO , Pb(NO,) 2 1.2 1 ±0.4 1

1.3 6 ± 0.3 7 1.7 1 ± 0.2 2 0.7 6 ± 0.0 4

0.6 5 ± 0.0 9 0.8 2 ± 0.1 4 8. 2 ± 1. 5

8. 0 ± 2. 5 58. 0 ± 3.0 * 2. 2 ± 0. 8

3. 3 ± 0. 9 2. 5 ± 0. 4 §Urin e wer e collecte d fo r 6 h r fro m rat s i n metaboli c cages . Student' s r-Test : *p<0.01 .P. 7

50 4 

### BARALDI £ 7 AL .

TABL E 9 CONCENTRATIONS (MEAN * S.E.M. ) OF ZIN C AN D COPPE RI N PLASM A AN D O F LEAD I

N WHOL E BLOO D OF PUPS (AGED 4 0 DAYS ) NOURISHED B

Y LEAD-POISONE

D MOTHER

S ( I mg/kg/day/os) TIL L WEANIN G AN D EXPOSE D FO R1 0 DAY S T

O LEA D ( I mg/kg/DA

Y B Y ORA L INTUBATION) co*w * CALCIU M

Treatmen t wate r NaNO , P1XNO,) , Plasm a (Mg/10 0 ml ) Zin c Coppe

r 120. 1 ± 3. 0 162.5 ± 10. 3 119. 8 ± 1.

2 161.

5 ± 15. 9 156.6 ± 11.7 * 179.0 ± 24. 2 Whol e Blood (Mg/10 0 ml ) Lea d 2.27 + 0.1 5 1.6 7 + 0.3 2 5.15 + 1.0 * 

### Student' s /-Test : *p<0.05 . 

# 5

• M.'A'.'.'JIL— * inniiiiir^* * o » 4 • i a • inaano * » « •otooitoxnnoiDoa o

yafr dr y waig M 

### FIG . 1 . Lead , zinc, coppe

r an d calciu m concentration

s i n brai n area s o f bab y rat s (4 0 day s o f age ) nourishe d b y lea d poisone d mother s ( I mg/kg/day/os

) an d treate d ( I mg/kg/day/os

) fo r 1 0 day s afte r weaning . Control s wer e expose d t o wate r o r NaNO :l 

### . Th e col umn s represen tth e mea n value s (±S.E.M. ) o

f 8 determination

s don e i n triplicate . Th e concentration

s ar e expresse d a s ftg/ g o f dr y weight . Black , whit e an d dashe d column s represen tth e value s o f water , NaNO , an

d P b (NO,).. , treate d rats,.respectively . Student' s /-test : *p<0.0

5 a tleas tvs . wate r an d NaNO, treate

d rats.
TABL E 10 META L CONCENTRATIONS I N WHOL E BRAIN S OF 60 DA

Y OLD RATS PR

E AN D POSTNATALL

Y EXPOSE

D T O LEAD ( 1 mg/kg/day/os ) 

### P b Z

n C

u C

a Wate r NaNO , Pb(NO,)

2 

### 0.3

5 ± 0. 5 0.3 7 ± 0. 8 1.4 2 ± 0.4 * 62. 2 ±6. 1

66. 8 ± 4. 2 55. 6 ± 3. 3 11. 9 ± 1.3

9. 8 ± 2. 1 11. 5 ± 1. 8 201. 8 ± 7. 3

193. 5 ± 6. 2 195.9 + 10. 1 

### Mea n ± S.E.M. ar

e expresse d a s jug/ g dr y weight . Student's/-Test : *p<0.001 .

u s t o tes tothe r brai

n areas : th e GAB A binding wa s foun d t o be reduce d als o i n the brainste

m an d i n the cerebellu

m bu tno t i n th e hippocampus . Althoug h i tseem s impossibl e fro

m the presentdat

a t o giv e a complete explanatio n o f the mech anism whereb y th e lo w affinit y GAB A receptors ar

e affected b y lead exposure , w e ca

n tentativel y suggest , i

n lin e wit h the hypothesi s o f

Silberged an

d Lamon [74] , tha

ta lead-induced increase d presenc e in th e brai n o f delta-aminolevulini c aci d coul d b e responsibl e fo r thi s down-regulation . I n addition , o r alternatively , w e ca n sugges tthatth e increase d presenc e o f zinc , whic h ha s recentl y been shown mainl y t o modulat e lo w affinit y GAB A receptors [6] , could , a tleas tin part

, b e re sponsibl e for thi s change i n GABA

A 

### recepto r characteristics . The increase d presenc e o f calciu m i n th e brai n tissue s doe s notsee m t o pla

y a rol e in thi s phenomenon , sinc e calcium ha s bee n describe d [21,49 ] a s modulating GABAA 

### hig h affin it y binding sites , a

n effec twhic

h requires , t

o b e optimized , a n incubation temperatur

e o f 37C 

### C , whic

h was no tused i n the presentexperiments . Thoug h thes e hypothese s nee

d t o b e born e outb y furthe

r experiments , w

e ma y conclud e fro

m the presentdat

a tha tther e i s a lowe r functiona

l activit y o f th e inhibitor y GABAergi c syste m i

n lead-expose d rat

s in com parison wit

h controls . Study o f the binding characteristic s o f dopamin e recep tors i n th e striatu

m of lead-expose d rat

s a t4 0 day s of age usin g :,

### H-spiperon e a s a ligan d and (-)sulpirid e a s a bindin g displacer , a s previousl y describe d [4] , showe d tha tther e i s a modestl y increase d affinit y o f th e dopamine receptors labele d b y :l

### H-spiperon e (mainl

y D 2 

### type ) a s expresse d by a reduced valu e o f

th e K „ withoutan

y chang e i

n th e maximu

m numbe r (B max 

### ) o f

binding site s (Fig . 2) i

n comparison wit

h controls . Fro

m thi s finding we ma

y surmis e tha

tth e lo w leve l o f lead exposur e use d her

e (25 ppm

) ma y induc e a modes t chroni c reduction o f

dopamine i

n th e synapti c cleft , whic

h i n tur n migh texplai n the increase d affinit y o f dopamine recep tors . Thi s hypothesi s i s presentl y unde r investigatio n by measurin g the neurochemica

l correlate s o f

th e dopamin

e re cepto r study i

n ou r model . Preliminar y dat a fro m thes e studie s indicat

e tha tdopamine synthesi s i s reduced i n the striatu m o f ou r lead expose d rats . Thes e dat a see m t o b e i n lin e wit h th e previousl y reported reduction o f dopamin e re leas e [80 ] i

n th e corpu s striatu

m induced by chroni c expo sur e t o lo w concentration s o f PbCO., (40 ppm

) an d o f dopamine synthesi s [51 ] induced i n a dose-relate d manne

r b y lead exposur e (21 6 an

d 135 0 ppm) . I tha s been suggeste d [80 ] tha

tth e inhibitor

y effectoflea dP. 8

LOW-LEVE L LEA D NEUROTOXICIT Y 50 5

t | TABL E 1 1

•H-GAB A BINDIN G KINETI C CHARACTERISTIC S I N CORTICA

L SYNAPTI C MEMBRANES O F PUPS (4 0 DAY S O F AGE) NOURISHED B Y LEAD-POISONE

D MOTHE

RRATS DURING PR

E AN D POSTNATA

L LIF E AND EXPOSE

D TO LEA D FO

R1 0 DAY S AFTE

R WEANIN G (MEAN ± SD ) Treatmen t ( 1 mg/kg / day/os ) wate r NaNO , Pi-XNO,).;) •H35. 7 ± 0. 5 35. 2 ± 0. 7 38. 8 ± 0. 8 GAB A bindin g characteristics * B na x 1 K|)

2 B max 2 0.625 ± 0.02 280 + 2

4 3. 5 + 0. 4 0.60 0 ± 0.0 4 304 + 1

2 3. 3 ± 0. 3 0.65 0 ± 0.05 28

2 + 1 8 1. 7 ± 0 . I § *K„=dissociatio n constanti n nM. B m ,lx =maximum numbe

r o f bindin g site s i n pmol/m

g protein . §Student' s /-Test : p<0.0l.

o n dopamin e releas e coul d b e du e t o competitio n betwee n lea d an d calcium . Lea d ha s bee n show n t o decreas e th e cal ciu m uptak e o f

brai n mitochondria , a n effec twhic h lead s t o accumulatio n o f

fre e intracellula r calciu m [29] . Th e presen t demonstratio n tha tther e i s indee d a n accumulatio n o f cal ciu m i n th e brai n tissue s o f lead-expose d rats , albei tonl y indicativ e sinc e th e metabolis m o f th e differen tpool s o f

cal ciu m i s ver y complicated , woul d see m t o giv e suppor tt o th e abov e mentione d hypothesis . W e hav e no tye tperforme d a challeng e wit h dopaminomimeti c drug s o n ou r lead-expose d rats , bu ti ti s noteworth y tha tWinde r et al. [81 ] hav e re porte d increase d responsivenes s t o stimulatio n o f locomoto r activit y b y apomorphin e an d onl y margina l change s wit h amphetamin e i n rat s expose d t o a relativel y lo w leve l oflea d (30 0 ppm) . Thi s findin g seem s t o giv e credi tt o th e hypothesi s tha tth e increase d affinit y o f post-junctiona l dopamin e recep tor s coul d b e du e t o a lead-dependen tpartia l impairmen to f dopamin e turnove r a tth e pre-junctiona l level . Th e spontaneou s locomoto r hyperactivit y observe d coul d b e explaine d b y th e demonstratio n give n b y Shaywit z et al, [69 ] tha tpartia l destructio n o f DA-containin g neuron s i n neonata l rat s lead s t o hyperactivit y whic h appear s betwee n 1 2 an d 2 2 day s o f

age . In summary , take n togethe r thes e biochemica l findings see m t o indicat e tha ta modes timbalanc e betwee n increase d functiona l activit y o f

th e striata l postsynapti c dopamin e re ceptor s an d decrease d activit y o f inhibitor y GABAergi c sys te m develop s durin g lea d intoxicatio n whic h coul d explai n th e border-lin e behaviora l effect s describe d i n thi s study . Interestingl y Mem o et

al. [51 ] an d Govon i et

al. [32] , wh o originall y reporte d a n increase d presenc e o f Met-enkephali n i n th e striatu m o f

lead-expose d rats , hav e suggeste d tha tth e effec to f lea d o n nigro-striata l neuron s coul d b e a conse quenc e o f

othe r lead-induce d neurotransmitte r alterations , i.e. , th e striata l opiatergi c system . Enkephalins , whic h ar e presen ti n th e striatu m [36] , hav e i n fac tbee n postulate d a s inhibitor y modulator s o f dopamin e releas e [26 , 35 , 63] . Thoug h ontogeni c studie s o f th e derangemen to f

th e develop in g opiatergi c an d dopaminergi c system s i n lead-intoxicate d rat s wil l solv e th e proble m o f priority , a tthi s stag e o f ou r knowledg e w e ca n als o hypothesiz e tha tlea d induce d altera tion s o f th e dopaminergi c syste m coul d b e responsibl e fo r th e increas e i n Met-enkephali n b y analog y wit h simila r phenomen a describe d b y Hon g et al. [35] . Th e increas e i n Met-enkephali n i n lead-expose d rat s togethe r wit h th e pro - 'H-SPIPERON E >p«clll c bindin g (fmoJ/m g prat. ) FIG . 2 . Scatchar

d ploto f 'H-spiperon e bindin

g t o striata

l mem brane s o f pup s (40 day s o f

age ) nourishe

d b y lead-poisone d mother

s ( i mg/kg/day/os ) an

d treate d wit h lea d ( 1 mg/kg/day/os ) fo

r 1 0 day s afte r weaning . Controls were expose d t o a n equivalenttreatmen

t wit h NaNO , o r water . Th

e value s (mean+S.E.M. ) o f th e affinit

y constant(K„) expresse d a s nM, an d o f the maximu

m numbe r o f bindin g site s (B max ) expresse d a s fmol/m g prot. were : Wate

r (• ) K„=0.18±0.02 , B max =148±4 ; NaNO , (• ) K„=0.18±0.03 , B ma ,, = 162+5 ; P

b (NO,) 2 (A ) K D =0.08±0.0 2 (a) , B m „ = 150±4 . Stu dent' s /-test : (a)p<0.0 1 vs . water an d NaNO, .
TABL E 1 2 /S-ENDORPHI

N CONCENTRATION

S IN PITUITAR

Y LOBES OF PUP

S (4 0 DAY S O F AGE

) NOURISHE

D B Y LEAD-POISONE

D MOTHER

S AN D TREATE

D ( 1 mg/kg/day/os) FO

R1 0 DAY S AFTE

RWEANING (MEAN + S.D. )

Treatmen t Wate r NaNO , Pb(NO,) 2 ^-Endorphi n (ng/gland) Anterior Neurointermediat e lob e lob e 520. 3 + 31. 5 803. 4 ± 63. 5 585.8 + 58. 1 1043. 6 ±239. 1 1360. 9 + 128.2 * 2187. 8 + 320.2 * Student' s /-Test : */><0.001 .

TABL E 1 3 HYPOTHALAMIC 0-ENDORPHI N AN D MET-ENKEPHALI N CONCENTRATIONS O

F PUP S (40 DAY S OF AGE) NOURISHE D B Y LEAD-POISONE

D MOTHER

S AND TREATE

D ( 1 mg/kg/day/os) FO

R1 0 DAY S AFTE

RWEANING (MEAN * S.E.M. )
Treatmen t /3-Endorphi n (ng/m g prot. ) Met-Enkephali n (ng/m g prot. ) Wate r NaNO , Pb(NO,) 2 36. 0 + 5.2

24. 3 + 6. 8 10.5 + 3.5 * 6.43 + 1. 0 5.8 4 + 1. 3 11.50 ± 2.4 * Student's/-Test : *p<0.01 .

### P. 9

50 6 BARALD I ETAL. TABL E 1 4 INCREASE D ANTINOCICEPTIVE RESPONSE S TO MORPHIN E ADMINISTRATIO N O F PUP S (4 0 DAY S O F AGE ) NOURISHE D B Y LEAD-POISONE D MOTHER S AN D TREATE D ( I mg/kg/day/os ) FOR1 0 DAY S AFTE RWEANIN G Chroni c pretreatmen

t Wate r NaNO , PtXN0 3 ) 2 Reactio n time § in se c

0 2. 8 + 0. 2 3. 4 + 0. 3 4. 2 ± 0.5 * Treatment :

Morphin e ( 5 mg/k g IP ) Morphin e ( 5 mg/k g IP ) Morphin e ( 5 mg/k g IP ) (mea n ± S.E.M. ) 3 0 4. 0 ± 0. 4 5. 2 + 0.5 * 7. 2 ±0.l t at : 6 0 mi

n 3. 2 + 0.0 9 4. 7 ± 0.3 * 5. 9 ± 0.5 * 

### §Usin g ho tplat e tes t(5 6 + 0.5°C). Student's/-Test : *p<0.05 , tp<0.001 .

posal tha tthi s peptid e ma

y mediat e euphoric state s [10 ] an

d facilitat e maze performanc

e i n rat s [40] , suggeste d t o u s tha t w e tes tth e functiona

l statu s o f

opiat e receptors an

d assa y th e leve l o f

endogenou s opiate s in th e hypothalamus an d pituitary . A s shown in Fig . 2 , we foun d a dramati

c increas e o f opiate receptors labele d b y 3 

### H-naloxon e in th e hypothal amu s o f lead-expose d rat

s (a t4 0 day s o f

age ) in compariso n wit h th e tw o control groups . The 3 

### H-naloxon e binding study wa s performe

d in brai n membran

e preparations a s previ ousl y describe d [3] . The numbe r o f binding sites , bu

tnotth e affinit y constant , o f

th e lead treated group wa s significantly highe r tha

n in th e cas e o f

group s o f rat s treated wit

h NaN0 3 

### o r water . Furthermore , increase d 3 

### H-naloxon e bindin g wa s als o foun d in th e brainste

m an d striatu m (dat a notshown) . Immunoreactive 0-endorphin (ir-/3-END ) an d Metenkephalin e (ir-ME) concentration s determined a tth e sam e ag e i n th e medio-basa

l hypothalamus (MBH) , followin g th e extractio n procedur

e describe d by Rossie r et

al. [67 ] an

d th e radioimmunoassa

y previousl y describe d b y on e o f u s [61] , wer e foun d t o b e significantly lowe r an d higher respectively , in lead-expose d rat

s tha n in bot h group s o f

control s (Table 12) . A n explanatio n for suc h a reduced presenc e o f

ir-/3-EN D in th e MBH , whic

h contain s th e cel l bodie s where thi s pep tid e i s synthesised , ma

y b e give n b y th e finding (Table 13 ) tha tth e level s o f

ir-i3-EN D ar e markedl

y increase d in th e anterio r an

d neurointermediat

e lobe s o f th e pituitar

y o f th e lead-expose d rats . Iti s likely , i n fact , tha

ta blockad e o f /3-endorphi

n releas e fro

m th e pituitar

y directl y o r indirectly elicite d by lead coul d b e responsibl e throug h activatio n o f

a feed-bac k proces s o f

reduced synthesi s o f

/3-endorphi

n in th e MBH . A mor e complicate d explanatio n o f th e reduced presenc e o f ir-/3-EN D in th e MB H coul d b e tha ta lead induced chroni c increas e o f

ir-M E (Table 12

) will trigger reduced dopamine releas e [37 ] an

d i n tur n a hyperprolactinemi c state [33,37] , whic h migh taffec tth e synthesi s of/3-endorphin in th e hypo thalamus [58] . Whatever th e mechanism b y whic h lea d in duce s thi s effect , th e chronically reduced leve l o f /3-endorphi

n i n the hypothalamus an

d probabl y i n othe r brai

n area s coul d induce a supersensitivit y phenomeno n a

tth e opiat e recepto r level . Othe r factors , however , ma

y be considered a s responsibl e fo r thes e abnormalities i n endogenou s opiat

e level s an

d opiate receptors characteristics . Th e lead-induced increas e 20 0 30 0 40 0 so o ao o *H-MALOXON

E f-Mdfl c MmMn g ifmol/mf l p«*o*- l FIG . 3 . Scatchar

d plo tanalysi s o f 3 

### H-naloxon e bindin g t o hypothalami c membrane

s o f pup s (4 0 day s o f age ) nourishe d b y leadpoisone d mother s ( 1 mg/kg/day/os

) an d treate d ( 1 mg/kg/day/os

) fo r1 0 day s afte r weaning . Control s wer e expose d t o a n equivalen

ttreatmen twit h NaNO, o

r water . Th e value s (mean±S.E.M. ) o

f th e maximu m numbe r o f bindin g sites (B ma 

### ,) , expresse

d a s fmol/m g protein , an d o f th e affinit y constan t(K„) , expresse

d a s nM , were : wate r (Q ) K o 

### =2.8±0.3 , B ma



### »=272+U.2 ; NaNO, (A

) K D 

### =4.2±0. 4 (a) , B m 

### „=373+12. 3 (a) ; P

b (NO,)2 

### (O

) K D 

### =2.3±0.5 , B m



### „ = 670+14. 6 (b) . Student' s /-test : (a)p<0.0 5 vs . water ; (b)p<0.00 1 vs . wate r an d vs . NaNO,.

o f brai n concentration s o f

zin c an d calcium i

n th e early stage s o f th e lif e describe d above , coul d pla y a rol e i n thes e abnormalities . Bot

h zin c an d calcium have bee n shown t o exer ti n vitro [7,75 ] an

d i n viv o [7,68 ] a

n antinarcoti c effect by interfering wit

h th e bindin

g capacit y o f opiat e receptors and/o r othe r factors suc h a s fragment s o f /3-lipotropi

n (i.e. , a-melanocyte-stimulatin g hormone , adrenocorticotropi c hormone an

d its fragments) , whic

h hav e affinit

y for brai

nP.1 0

LOW-LEVE L LEA D NEUROTOXICIT Y opiat e receptor s an d inhibi tmorphine-induce d analgesi a [11 , 20 , 26 , 38] . Moreover , no tonl y lea d [56 ] bu tals o zin c [1 ] an d calciu m [62 ] ion s hav e bee n show n t o b e implicate d i n th e regulatio n o f

th e cerebra l activit y o f adenylat e cyclase , a n enzym e whic h ha s bee n suggeste d t o pla y a rol e in th e de pendenc e an d toleranc e t o chroni c morphin e administratio n [70] . Th e abov e mentione d hypersensitivit y o f th e opiat e re ceptor s induce d b y lea d exposur e wa s prove d t o b e operativ e i n "i n vivo " experiment s b y performin g a ho tplat e test . A s show n i n Tabl e 13 , th e lead-treate d rats ha d a longe r reactio n tim e an d wer e supersensitiv e t o th e administratio n o f a threshol d dos e o f

morphine . Incidentally , a n increase d re spons e t o morphin e administratio n o f lead-expose d rat s ha s bee n reporte d b y other s whil e this pape r wa s i n preparatio n [41] . j I tmus tb e mentione d that , whil e th e chroni c treatmen t wit h NaNO. , di d no tchang e th e bindin g characteristic s o f th e dopaminergi c an d GABAergi c receptors , i twa s abl e t o in duc e a wea k b y statisticall y significan tincreas e i n th e numbe r o f opiat e receptors , whic h parallele d a sligh tdecreas e i n /3-i:ndorphin , increase d reactio n tim e i n th e ho tplat e tes tan d a n increase d respons e t o a threshol d dos e o f morphine . 50 7
Thes e dat a see m t o indicat e that , atleas tfo r th e opiatergi c system , NaNO. , i s no ta completel y iner tcompound . Thi s dat a i s no tcompletel y surprisin g sinc e a n increase d presenc e o f

Na 4 ha s bee n show n t o induc e majo r change s i n th e bindin g t o opiat e receptor s [60] . Since , however , ther e ar e n o othe r dat a availabl e o n th e effec to f a perinata l chroni c lo w exposur e t o NaN0 3 o n th e opiatergi c system , thi s NaN0 3 -induce d effec tneed s t o b e furthe r clarifie d experi mentally . Itmust , however , b e stresse d tha tsuc h a sligh teffec to f NaN0 3 doe s no tmarkedl y interfer e i n th e P b (N0 3 ) 2 - induce d effec tsinc e th e numbe r o f opiat e receptor s foun d i n lea d treate d rats i s statisticall y highe r i n compariso n no tonl y wit h th e wate r bu tals o wit h th e NaN0 3 treate d rats. T o conclude , th e firstobservatio n reporte d her e o f a supersensitivit y o f

th e opiatergi c syste m whic h mediate s pai n perception , i n paralle l wit h a n increase d presenc e o f Met-enkephali n i n th e hypothalami c are a whic h migh taffec t directl y o r indirectl y moto r functions , seem s t o b e th e mos t promisin g dat a o f

ou r presen tstudy . Thes e data , i n fact , i f confirme d b y furthe r experiments , ma y provid e a ne w in sigh ti n th e understandin g o f th e comple x mechanis m oflea d neurotoxicity . ACKNOWLEDGEMEN

T Thi s researc

h was supporte

d b y Italia n C.N.R . gran

t(No . 8302976-56) .
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