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# menta l Kniironmrntul Health Pemp^eiiies Vol. I2,pp. lt'J-123, 1975 

# am d Beiia^sor a s m the Rat e to Lea d durin q Chroni c i 

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# b y Lawrenc e W . Reiter, * Georg e E . Anderson, * Joh n W . Laskey, * an d Danie l F . Cahill *

Youn g mal e an d femul e Sprague-Dawle y rat s wer e give n drinkin g wate rcontainin g 5 o r 5 0 pp m P h fo r1 0 day s prio rt o mating . Pregnan tfemale s wer e continue d o n thes e regimen s throughou tgestatio n an d lactation . Afte rweanin g th e offsprin g wer e similarl y expose d throug h adulthood . Kefle x development , bod y weights , an d locomoto ractivit y wer e measure d i n th e offspring . Significan tdelay s wer e note d i n th e developmen tofth e right in g refle x a t5 an d 5 0 pp m an d i n ey e openin g a t5 0 ppm . N o differenc e wa s observe d i n developmen to f

th e startl e refle x a teithe rdose . Mea n bod y weight s o f treatmen tgroup s durin g thi. s developmenta l perio d wer e no tsignificantl y differen tfro m controls . Locomo to ractivit y wa s measure d i n adul tmale s utilizin g aresidentia l ma/e . Mot h level s oflea d produce d asignifican treductio n i n locomoto ractivity . Whe n group s wer e treate d wit h (Iamphetamin e (1. 0 mg/k g subcutaneous) , lea d treatmen tcause d adose-relate d diminutio n i n th e amphetamine-induce d hyperactivity . Thes e result s indicat e tha trat s expose d t o lo w level s o f lea d fro m conceptio n unti l adulthoo d sho w adela y i n nervou s syste m development . A s adults , thes e animal s exhibi t hypoactivit y an d decrease d responsivenes s t o amphetamine . 

## Introductio n Loa dtoxicosi s i nchildre nha s bee n implicate di n hyperkinesi s (/ ) an dmenta l retardatio n(2) . Thes e finding s hav e raise dth e possibilit y tha t chronic , low-leve l exposur e t o lea dma y produc e "silent " brui n damage . Anima l experiments , usin g avariet y of species , hav e demonstrate dtha t perinata l ex posur e t o lea dproduce s simila r behaviora l change s includin g hyperactivity , periphera l ataxia , an dag gressivenes s (-J-5) . Thes e experiments , however , hav e employe ddietar y level s o f lea drangin g fro m 120 0 t o 27,00 0 ppm . Th e presen t

stud y wa s undertake nt o tes t for possibl e developmenta l an dbehaviora l change s produce db y continuou s pre - an dpostnata l ex posur e t o lo w level s of lead . Th e result s indicat e lha t behaviora l change s ca nb e produce db y level s a s lo w a s 5 pp mi ntb e diet .

' K\|a - - l iiiirtit.i l I ti i il t >|*. > Laboratory . Nationa l Kiivironnn- n I a l Ui'-M-'ircl i Center , U.S . Kiivii'oimiriita l Protectio n Agency , It . -.-..l.-l i Triancl e Park . N.< \ L>77 1 I . Decembe r197 5 

## Method s Mal e an dfemal e Sprague-Dawle y rats , obtaine d fro mBlu e Spruc e Farm s (Altamont , N.Y.) , wer e randoml y assigne dt o on e o f thre e treatmen t groups : 0 , 5 , o r

5 0 pp mlea dadministere di nth e drinkin g wate r

i nth e acetat e form . Animal s wer e pretreate dfo r

aperio do f

4 0 days , whic hallowe d lea dconcentration s t o approac hsteady-stat e level s i nsof t tissue s (6). Followin g pretreatment , animal s wer e mate dan dpregnan t female s wer e continue d o nlea dthroughou t

gestatio nan dlactation . A t weaning , th e offsprin g wer e similarl y expose d throug h180 days . Thes e F i generatio nrat s wer e mate da t

9 0 day s o f

age , an dth e resultin g F j generatio nanimal s wer e expose dt o th e sam e lea d regimen . Fiv e pup s fro meac ho f 4- Glitter s pe r

treatmen t grou p inbot hth e F i an dFageneration s wer e teste d fo r th e age s a t

developmen t

o f th e startl e response , tb e rightin g refle x an dth e ag e o f ey e opening . Th e startl e respons e wa s evoke db y aclic k (generate d b y ato y clicker ) sounde dimmediatel y behin dth e ll« > i



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hea do f a n anima l hel dsuspende db y th e nap e o f th e neck . A positiv e respons e consiste dof amuscl e jer k i n an y o f

th e extremities . Th e rightin g refle x wa s elicite db y holdin g th e ra t upsid e dow nb y th e nap e ofth e nec k an dth e tai l an ddroppin g i t

fro mapproximatel y 3 0 c mabov e som e woo dshavings . A respons e wa s considere d positiv e if

th e anima l righte di nmid-ai r an dlande d o n al l fou r

feet . Ag e a t ey e openin g wa s recorde dwit hcomplet e openin g o f

bot heyes . Fiv e pup s fro meac ho f 9-1 1 litter s (F i + Fa)pe r treatmen t

grou p wer e teste ddail y betwee n8:3 0 A.M . an d10:3 0 A.M . Th e litte r mean s wer e use dt o calculat e th e mea nda y o f developmen t

o f eac h response , fo r

eac htreatmen t

group . Locomoto r

activit y o f

adul t male s wa s recorde d a t approximatel y 12 0 day s o f ag e i naresidentia l ma/ e (Fig . 1 ) describe dinitiall y b y Norto n e t al . (7) . Group s o f

thre e littermate s wer e teste di nth e maz e fo r

aperio do f

5 days . Fou r

maze s wer e house dinasound-attenuate droo mmaintaine do n a1 2 h r light/1 2 h r

dar k cycl e beginnin g a t

6:0 0 A.M . Foo dan dwate r wer e availabl e ad

libitum. Th e dail y experimenta l procedur e wa s a s follows : Group s wer e introduce dint o th e maze s a t 10:0 0 A.M . an dremove da t

9:0 0 A.M . th e followin g day , th e maze s wer e cleaned , foo dan dwate r wer e replenished , an dth e animal s wer e the n reintroduce dint o th e maze s a t 10:0 0 A.M . Activit y count s wer e recorde da t 1-hr interval s b y utilizin g a Xero x minicomputer .

Kna>i(i- : I Schemati c representatio n o f th e i'i-siileiiti.'i l maz e UM-< Il o measur e locomoto r activity . Tli e maz e <on-ist s of a serie s o l iiiierconncrlci l alley s 11 0 c mwide . Il l c mhigh ) con vergin g o nt

i centra l upenfiel dan dcovere dwit htransparen t acryli c plasti c equippe dwit hacentra l raise dare aove r th e ope nfield . Th e overal l dimension s ar e 7li c mwid e x (i O c m deep . I.on.mutto n i s detecte db y eigh t phototrarisisto r ( + ('infrare dlight-emittin g diod e (o l pairs . 12 0 JA'«a-WlM>VWW.t M l '••••-•••»' « fMW| J &m

Maz e activit y wa s als o recorde dfollowin g th e administratio no f

(/-amphetamin e HC l (4'rng/kg ) administere dsubcutaneousl y (SC ) o nda y 5 , 2 0 mi n prio r t o reintroducingjhejinimal s int o th e maze . Grou p means , fo r

both'refle x developmen t an d locomoto r

activity , wer e compare db y tisin g th e ttest . 

## Result s N o difference s betwee nth e F i an dF 2 genera tion s wer e observe di nan y paramete r an dresult s fo r bot hgeneration s hav e bee ncombined . Th e effect s o f

lea dtreatmen t

o ndevelopmen t ar e show ni nTabl e 1 . Developmen t o f th e startl e response , whic hoccur s normall y a t 1 2 day s o f

age , wa s no t significantl y altere db y lea dtreatment . O n th e othe r hand , bot hey e openin g an ddevelopmen t o f th e rightin g refle x wer e significantl y delaye d(p < 0.05 ) b y lea dadministration . Ey e openin g wa s delaye dapproximatel y 1 da y b y th o 5 0 pp mtreat ment , wherea s th e rightin g refle x wa s delaye d1 da y b y 5 pp man d2 day s b y 5 0 ppm . Tabl e 1 . Effect s oflea dadministratio n o n refle x develop

men t an dey e openin g i n th e rat . P b dose , ppm " 0 5 5 0 No . o f litter ; .\ * 1 1 ! i 1 0 Mea n da v

Startl e 1 l.- S + 0. 2 12. 0 ± 0. 2 12. 4 ± 0. 2 n f

developmen t

± S.K . t y e openin g 15. 2 + 0. 2 15. 1 ± 0. 2 |5.H±0.2' ' Kightin g

17. 7 i 0. 2 18. 8 ±0.-1' ' 19. 8 + 0.7 '

a [)os e gi v e n a ; pp mo f

lea di n th e drinkin g water . * W~ numbe r

o t litter s ( 5 aniniaU'lirter ) ''Significantl y differen t

fro mcontrol , r-test,/ > < 0.05 . Bod y weight s fo r

mal e rat s durin g thi s perio do f developmenta l testin g ar e show ni nFigur e 2 . N o consisten t

difference s i nbod y weight s o f th e treat men t group s wer e observed , althoug hth e 5 0 pp m lea dgrou p wa s significantl y heavie r tha ncontrol s o nday s 1 5 an d18 . Female s showe dsimila r growt h curves . Figur e 3 show s representativ e 23-h r time-inter

va l histogram s fo r

th e variou s treatmen t group s take no nda y 3 i nth e maze . Animal s showe da ninitia l hig hleve l o f ex plorator y activit y durin g th e firs t hou r inth e maze . Followin g thi s initia l perio do f exploration , activit y showe danorma l circadia nrhyth mwit hlo w diur

na l (photolight ) activit y an dhig hnocturna l (photodark ) activity . Dat aobtaine ddurin g thes e variou s periods , i.e. , exploratory , diurnal , an dnoc turna l a s wel l a s tota l activit y wer e analyze dfo r lea deffect s

Environmenta l Healt hPerspective s 

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Kl(a.'U K 2 . Growt hcurve s fo r contro l an dlead-treate dmal e rat s durin g th e perio do f developmenta l testing:(» ) control ; (A ) 5 pp mPb ; (n ) 5 0 pp mPb . Kac hpoin t represent s th e mea n( ± S.E. ) fo r

1 2 animals . Th e compile ddat afo r maz e activit y o nth e first fou r successiv e day s of

testin g ar e presente di n Figur e 4 . Activit y o nth e firs t cla y i nth e maz e wa s highe r tha n o nsubsequen t

days . Thi s differenc e wa s observe di nal l treatmen t group s an ddurin g al l tim e periods . Thi s elevate dactivit y represent s th e animal' s increase dreactivit y du e t o th e nove l environment . B y th e secon dday , th e animal s becam e "established " i nth e maz e an dsubsequen t activit y level s wer e consisten t fro mda y t o day . Lea dadministratio nproduce dasignifican t (p < 0.05 ) decreas e i n locomoto r activit y rangin g fro m 2 7 t o 42 % o nda y 1 an dfro m1 1 t o 37 % o nda y 4 . Thi s lead-induce dhypoactivit y wa s no t dose-rel

ated . Bot htota l an dnocturna l activit y remaine d depresse di n bot hlead-treate dgroup s throughou t th e 4-da y period . B y da y 4 , neithe r lead-treate d grou p differe dfro mcontro l durin g th e explorator y perio dan dfollowin g th e firs t da y n o grou p difference s wer e foun di nth e diurna l period . Twent y minute s prio r t o testin g o nda y 5 , animal s wer e give nsubcutaneou s (SC ) injection s o f 4. 0 mg/k g {/-amphetamin e HCl . Th e result s pre sente di nFigur e 5 represen t th e chang e i nactivit y (A ) followin g amphetamin e administration . Th e A activit y was.determine db y subtractin g tb e activit y obtaine do nda y 4 (predrug , contro l levels ) fro mth e Decembe r

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KICIIK K 3 . Representativ e time-interva l histogram s fro mcon tro l an dlead-treate dani:r.als . Dat awa s obtaine do n da y 3 i n th e maz e frorr . group - of thre e adul t

males . Fo r

purpos e o f analysis , th e dat aha ? b^-er . divide dint o th e variou s activity

period s represente db y th e shade dareas . activit y followin g amphetamin e an dtherefor e represent s th e drug-induce dchange s i nactivity . I ncontro l animals , amphetamin e produce dth e expecte dhyperactivity . Sinc e dru g wa s ad

ministere ddurin g th e diurna l period , whe nactivit y i s normall y low , th e majo r effec t wa s see ndurin g thi s period . Activit y i ncontro l animal s wa s ele

vate dt o 444 ^ an d6.52' c of predru g levels , durin g th e explorator y an ddiurna l periods , respectively . Thi s wa s als o reflecte di na nelevatio no f tota l ac tivit y t o 224 % o f

predru g contro l level . Durin g th e nocturna l period , however , th e contro l activit y fel l t o 80 % o f

th e predru g level , demonstratin g apar tia l compensatio nfo r

th e drug-induce dhyperac

tivity . Lead-treate danimal s showe dadiminutio nin th e amphetamin e resjxins e (Fig . 5) . Whe ncom pare dt o th e ^ activit y of th e contro l group , bot h treatmen t

group s showe dasignifican t Ip < 0.05 ) decreas e i namphetamine-induce dactivity . Thi s decrease dresponsivenes s wa s als o observe dwhe n activit y wa s expresse da s percentag e o f predru g 12 1 ' 5

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4 1 DAY S I N MAZ E FICUU K 4 . Kffectso f

lea dadministratio n o n locomoto r activit y i n adul t

mal e rats : (• ) control ; (A ) 5 pp mPb ; (U ) 5 0 pp mPb . Animal s wer e teste di n group s o f three , an deac hpoin t represent s th e mea n (counts/h r +S.K. ) fo r

fiv e group s o f

animals . Dat aar e presente dfo r th e variou s activit y period s fo r th e firs t 4 day s i n th e maze . Point s marke d'"a " ar e significantly

differen t fro mcontro l (/-test,/ ) < 0.05) . levels . Wherea s control . activit y wa s elevate dt o 224 , 111 , an d(iS2 % durin g th e total , exploratory , an ddiurna l periods , respectively , th e 5 0 pp mgrou p wa s elevate dt o K3S , 323 , an d'.V.lS',r durin g thes e sam e periods . A s wit hth e controls , treatmen t group s showe dles s activit y durin g th e nocturna l perio dan dn o significan t difference s betwee n group s wer e presen t

durin g thi s period . 

## Discussio n Hal s continuousl y expose dfro mconceptio nt o lo w level s o f

lea dshowe dadela y i nnervou s syste m .developmen t

a s see nb y adela y inth e rightin g reflex . I'erinata l exposur e ha s previousl y bee n reporte dt o caus e delaye ddevelopmen t (N) , bu t a t dietar y level s whic hproduce dafal l infoo dcon sumptio nwit haconcomitan t retardatio no f growth . Thi s undernutrition , whic hi s know nt o dela y developmen t

(.9) , wa s sufficien t

t o produc e a simila r dela y i npair-fe dcontrols . I nth e presen t study , n o difference s i ngrowt hrate s wer e observe d durin g th e perio do f

refle x testin g whic hsuggest s tha t th e observe ddela y i ndevelopmen t wa s :h e resul t o f adirec t

effec t o f lea do nth e nervou s syste mo f

th e pups . Dail y locomoto r

activit y wa s depresse ddurin g fou r consecutiv e day s o f testin g i nadul t

lead

treate danimals . O nth e firs t tes t day , hypnactivit y wa s observe ddurin g al l period s ofth e dail y activit y cycle . However , o nsubsequen t day s n o difference s 12 2 Environmenta l Healt hPerspective s 

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5 5 0 0 5 OOS f t f A O I N U H INKIN G WAHH . pix n treate danimal s i s no w bein g teste di nou r labor

atory . Finally , th e observe dattenuatio ni nth e respons e o f lead-treate danimal s t o amphetamin e ad

ministratio nha s bee nconsistentl y observe dbot hi n mic e (15) an drat s (I'd). Furthermore , thi s diminishe dresponsivenes s ha s bee nobserve dwhe n baselin e activit y i s elevate d(15), norma l (13) o r depresse d(presen t

study ) b y lea dtreatment . 

## Acknowledgement s W e wis ht o than k

Ms . Miria mAs hfo r

he r tech

nica l assistanc e durin g al l phase s o f thi s work . KKKKKKNCK S f ' | ! 

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d-amphetamine administratio n (4. 0 m g kg ) o nlocomoto r

activit y o f

contro l an dlea dtreate drats . Value s ar e presente da s A activit y (counts/h r ± S.K. ) follow

in g amphetamine ; A activit y - (activit y o n dru g day ) - (ac

tivit y o n th e predru g day) . Kac hbar . therefore , represent s th e drug-indus-e dlocomoto r activit y fo r

five group s o f animals . Bar s marke d"a " ar e significantl y differen t fro m contro l (f-test. p < 0.05) . wer e observe di ndiurna l activit y an db y th e fourt h day . explorator y activit y (define da s th e firs t hou r in th e maze ) wa s als o a t contro l levels . Thi s tran sien t differenc e i nactivit y suggest s tha t lea dtreat men t alter s th e animal' s reactivit y t o anove l situa

tio nan dtha t

onc e stabl e activit y i s established , • •nly nocturna l activit y i s affecte db y lea dtreat ment . Simila r

variation s i nth e responsivenes s o f animal s t o administratio n o f othe r chemica l agent s fts afunctio n o f

tim e hav e bee ndescribe d(10). Tiii s findin g o f

hypoactivit y ha s adde dmor e fue l :•< th e fir e o f

confusio nwhic hconsume s th e lea d literature . Al l possibl e change s i nlocomoto r ac tivit y hav e bee nreporte dfollowin g lea dtreatment : hyperactivit y ('l.ll), hypoactivit y (12), an dfinally , r. o chang e i nactivit y (l'.l). I n th e presen t

study , animal s wer e teste da s adults . Th e possibilit y exist s tha t th e lea deffec t

o n •activity inth e ra t

i s age-dependen t

wit hinitia l hy

peractivit y i n youn g animal s (// ) followe db y hy

j'.activit y i n th e adult . Thi s tim e cours e woul db e • "iisisten t

wit hth e relationshi p of lea dtoxicosi s -villi minima l brai n dysfunctio ninchildre nsinc e •;it

- hyperactivit y inchildre ngenerall y disappear s •' . i' hage , th e so-calle d"maturationa l lag " (/'/) . Tri e seria l tim e cours e o f activit y change s i nlea d 1 . David , O. , e t al . Lea dan dhyperactivity , Lance t 2 : 90 0 (1972) . 2 . Beattie , A . D. , e t

al . Rol e o f

chroni c low-leve l lea dex posur e i n th e aetiolog y o f meta l retardation . Lance t 1 : 58 9 (1975) . 3 . Pentschew , A. , an dGarro , F . Lea dcncephalnmvc-lopath y o f th e sucklin g ra t

an dit s implication s o n th e porphyrinopathi c nervou s diseases . Act aNeuropath . (Berlin ) 6 : 26 6 <196S> . 4 . Silbergeld , K . K. , an dGoldberg , A . M . A lead-induce d behaviora l disorder . Lif e Sci . 13 : 127 5 (1973) . 5 . Allen , J . R , McWey . P . J. , an dSuomi , S . J . Pathobinlogica l an dbehaviora l effect s o f lea dintoxicatio n i n th e infan t Rhesu s monkey . Knviron . Healt hPerspect . 7 : 2'1 9 (19741 . 6 . Castellino , N. , an dAloj . S . Kinetic s o f distributio n an dex cretio n oflea di n th e rat . Brit . J . Ind . Med . 21 : 30 S 11964 i 7 . Norton , S. , Culver . B. . an dMullenix , P . Measurement s o f th e effect s o f

drug s o n activit y o f

permanen t

group s o f

rats . Psychopharmacol . Comm . 1:13 1 (1975) . 8 . Michaelson , I . A. , an dSauerhoff , M . VV . A nimprove d mode l o f

lead-induce dbrai n dysfunctio n i n th e sucklin g rat . Toxicol . Appl . Pharmacol . 28:8 8 (1974) . 9 . Smart , J . L. , an dIXibbing , J . Vulnerabilit y o f developin g brain . II . Kffect s o f

earl y nutritiona l deprivatio n c n refle x ontogen y an ddevelopmen t o f behaviou r

i n th e rat . Brai n Res . 28:"8 5 (1971) . 10 . Reinberg , A. , an dHalberg , F . Circadia n chronophar

macology . Ann . Rev . Pharmacol . 11 : 45 5 (1971) . 11 . Sauerhoff , M . VV.

, an dMichaelson , I . A . Hyperactivit y an d brai n catecholamine s i n lead-expose ddevelopin g rats . Scienc e 182 : 102 2 (1973) . 12 . McLellan , J . S. , e t al . Developmenta l toxicolog y o f lea di n th e mouse . Fed . Proc . 33 : Abstr . 47 9 (197-1) . 13 . Sobotka , T . J. , an dCook , M . P . Postnata l lea dacetat e ex posur e i n rats : Possibl e relationshi p t o minima l brai n dys

function . Amer . .1 . Ment . U.-fic . 79 : 5 (1971) . I-I . Wender , P . H . Minima l Itrai n Dysfunctio n i n Children . Wiley-lntcrscii'iuc , New York , 197 1 p . 75 . 15 . Silbergeld , K . K. , an dColdberg , A . M . I*ad-induce d behaviora l dysfunction : a n anima l mode l o f hyperactivity . Kxp . Neurol . 12 : lll i (1971) . 

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