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# 31)11-1-15 3 Docket Number: 

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### Toxi c Effect s o f Lea d o f Neurona l Developmen t and Functio i
Rober t Freedman , Lar s Olson , and BarryJ . Hoffe r 

# B 

# i e t n 

# f i YM 

# I 3 193 2 EP A AI R DOCKE

T Department s o f Psychiatryand Pharmacology , Denver VAM C L 

### ~a'rib ' uhiversit yo t Colorad o Healt h Science s Center , Denver , C O 8026 2 an d Departmen t o f Histology , Karolinsk a Institute , Stockholm , Swede n

Th e effect s o f lead o n th e developmen t o f the nervou s syste mar e o f immediat e concer n t o huma n welfare . Whil e i t i s clea r tha t lea d ca n affec t
neurona l developmen t a t level s o f exposur e withi n th e rang e foun d i n th e environment , th e particula r mechanis mo f the disruptio n i s no t easil y ascertained . Lac k o f knowledg e o f the mecrianis m o f damag e hamper s i t s treatmen t and prevention . Th e purpos e o f ou r researc h i s t o develo p a mode l syste mi n whic h th e effect s o f lead o n centra l nervous.systemdevelopmen t ca n b e demonstrated . Th e complexit yo f the brai n i s a barrie r t o suc h investigations , becaus e ofte n i t i s no t clea r i f apparen t toxi c effect s represen t change s secondaryt o somati c changes , suc h a s endocrin e o r hematologica l defect s tha t coul d affec t brai n development , o r eve n transneurona l effect s cause d b ytoxicit ya t a dista l sit e tha t depriv e a brai n are a o f a synapti c inpu t neede d fo r it s prope r development . Arelate dP. 3



### proble mi s th

a redundanc yo f compensatorysystem s i n th s brain . Suc h system s maydisguis e th e severit yo f th s initia l toxi c insul t and themselve s ca n caus e functiona l disturbances . W e hav e grafte d discret e brai n region s derive d from ra t fetuse s int o th s anterio r chambe r o f th e ey e o f adul t host s i n a n effor t t o stud

yneurona l developmen t i n a syste mtha t minimize s suc h difficulties . Th e brai n piece s continu e organotypi c developmen t i n the eye , bu t ar e isolate d frompossibl e problem s i n the developmen t o f th e endocrin e and othe r somati c systems , becaus e th e adul t hos t ha s thes e system s alread yfull ydeveloped . Similarly , toxi c effect s mediate d b yconnectin g brai n area s ar e eliminate d sinc e tha transplan t i s isolate d i n th s anterio r chambe r o f the eye . usin g thi s syste mw s hav e discovere d tha t lea d induce s a hypernoradrensrgi c innervatio n o f centra l nsrvou s syste mtissue . Th s increase d innarvatio n i s observe d no t onl ystructurally , bu t als o functionally . Sinc e norepinephrin e i s a n inhibitor

yneurotransmitter , th s ingrowt h result s i n a slowin g o f th e discharg e o f nsuron s tha t normall yreceiv e noradrensgi c innervation . Studie s o f oths r tissue s sugges t tha t increase d axona l growt h and innervatio n mayb e a gansra l proble mo f lead intoxicatio n tha t extend s beyond th e centra l adrenergi c syste mt o encompas s othe r brai n areas , a s wel l a s periphera l sympathsfci c systems . Hyperactivit ymigh t b e th s behaviora l consequenc e o f thss a alteration s i n nsurona l dsvelopment .P. 4

Fto . I . Examples o f

intraocula

rbrai n graft s a s observed In vivo a

tapproximatel

y lO x magnification : (a) Hippocampa

l graf tobtaine d fro m a fetu s wit

h a CR L o f 25 m m 6 months postgnftlog . Th e hos t anima l was maintaine

d o n I * lea

d acetate . Thes e graft

s show norma

l vascularization (several bloo d vessel s ca n b e see n o o graf

tsurface ) bu tslightly impaire

d volum e increas e a s compare

d t o controls , (b ) Oraf to f the developin g substanti

a nigr a are a obtained from a fetus wit h a CR L o f 2 4 m m 2 months afte

r graftin g t o hos tanima

l o n ta p water . I n contrastt o cortica

l graft s suc h as i n (a) , thes e brai

n ste m graft

s obtai n a final volume smallertha n a tgrafting . Whit

e are a i n uppe rpar ti s lightreflex .

•aya a 

# 1 -
err T " 

# J

s i > • > «*Ma e Fio . 2 . Lea

d levels i n bloo d (soli d line , Mg/liter

) sn d brai n IH situ (dotte

d line , mg/kg ) a

s a functio n o f varyin g duratio n o f exposur e to 1 % lea d acetat e i n drinkin g water. Point

s sho w mea n values , an

d bars , SEM .P. 5

CORTE X CEREBEU

I tNIr

tat. *• 03RTE X CSEBai l

r T * WB 8 '— \ . . i . i >*« S frioji Effect

s o f chroni c 1

% lea d aceut e o n growt

h ofth e cerebella ranlage In oeulo. Cerebella r graft s wer e take n from fetuses wit

h a CR L o f 11-1 2 m m correspondin

g to gestationa

l Day s 1 4 (a) , an d 15-17 . gestationa

l Day s 16-1 7 (b) . Graft

s fro m mor e immatur e donors grew t o large

rfinal size s (b) . Ther e were n o significan

teffects o f

lea d o n growt

h i n eithero f

Ih e tw o groups , n » 19-2 4 (a) an d 21-2 8 (b) .P. 6

Piout l 1 . Creeyi-violet-etaine d sectio n o f alead-treate d cerebella r graf tobtaine d from da y 1 4 of

gestatio n a

asee n afte rintnoeoia r maturation . Not e th e hig h degre e of organotypi c organisatio n Tw o fob s o f

cerebella rcorte x can b e see n wit h free surface s eov ere d b y plal membrane .P. 7



# fTmW^J }
10 0 m se c B . "*• 100 -
« 76 - o so :

7 5 mse c 

# C .

s o ae c FIGUR E 2 . Spontaneous discharge from a graf tPurkinj e cel l i

n a sodiu m acetate-treate d animal (A ) Actio n potentia l recor d pho l from th e oscilloscope , (fl ) Interspac e Interva l histogra m wit h prominen

tmode l pea k indicating regularit

y o f discharge . Abeds M • i s forful l scale . (ORatemete rrecor d again showin g sustaine d regulardischarge . phe d

I I *-*• * Aeeiat

o H Sodlu a Aoetat

e

FIOUB I S . Histogra

m showin g th e distribution o f spontaneous dis charg e rate s o f

Puridnj e neurons from lea d acetate-treate d an

d sodiu m acetate-treate d cerebellargrafts . There i s a marke d difference betwee n th e tw o group s whic h i s statisticall y significan t (p < 0.001) . Not

a tha tth e majorit

y o f ceU s encountered i n lead treate d grafts wer e suenL (• ) denote s cell s whic h wer e a U derive d from' on e graft , th e only lead-treate d graf tcontaining Purkmj e neurons wit

h sustained , spontaneous discharge .P.8



# ••

non-Injecte d 1 mo/Kg Control * PbA

e Smg/Kf l Malnourishe d Untreate

d PbA e Contro l FIGUR E 4 . Ba rgrap h ahowin g mea n discharg e rat e ± SE M o f cerebella rPurkmj o neuron e from animal s injecte d postnatall y wit h NaAc , 1 m g PbAc/kg , 8 m g PbAc/kg , and nonmjoctcd controls . I n addition , th e mean firing rate s rf Purkinj e Moron s from malnourishe d animal s and from animal s concomitantl y raise d i n normalize d litter s ar e illustrated . Th e rate s i n 8 m g PbAe/k g animal s ar o significantl y differen t from thos e o f

NaA c animal s (p < 0.01) .



# JP. 9

FIGUR E 6 . Effect o o f

ka d Injecte d int o A e anterio rchambe ro f th e ey e i n a8-p L volum e o n sympathatl s odreaargi s narve e i n th e ro tIris . Illustrate d ore fiuoreccenee mJa-ophotograph s from representativ e area s of iri s whol e mount s processe d fo rvtotAntlo n o f catwhoHamine

a neeordih g t o Fak k an d HiOarp . (/USliam-lnJecte d eye . Norma l densit y o f

adrenergi c groun d plexu s i n dilate ? ora s o f th e irio . » 114 . (fl ) Adraitargi e hyperinnervatk e U day s afte rinjectio n o f

lea d acetate . Ther e iaalmos tn o decreac ai n msoressisnc Q intensit y (Le. , coto ehohinlft s content ) c f

individua l nerv e fibers. A n Innervate d bloo d vesse l traverse s from uppe rlof tt o lowe rright, x 114 .P.1 0



# • N o Acetat e Contro l

P b Acetate •firontal Cortex

I20- , P b Acetate-Parieta l Co r te n

P b Acetate-Occipita l Corte x

* ) ps.Of t •P » O l FIGUR E 8 . Cortica

l noradrenergic varieoeitie a in animal

s treate d neonatally with lead - o rsodiu m aceute . Estimate s wer e mad e b y blin d observer s usin g Falck-HUlar p fluorescence histochemica l techniques . Not e elevatio n i n lead-treated animal s i n all thre e cortica l regions .