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## ^ AI RDOCKET, JLA REFFECT S O F LEA D 

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R . A . Goye r an

d J . F . Moor e Departmen t o f Patholog y Universit y

o f Nort hCarolin a a t Chape l Hil l

Th e particula r leve l o f exposur e t o lea d whic hresult s ina n advers e healc heffec t i s relate d t o man y factor s includin g "criti ca l orga ncontent, " rout e o f administratio nan d a numbe r o f meta boli c an

d nutritiona l factor s (Goye r and Mahaffey , 1972) . Impor tan t consideration s relate d t o toxi c manifestation s o f a heav y meta l ar e it s distributio namon g subcellula r component s an d inter actio nwit hcellula r functio nan d whethe r i t i s complexe d t o a diffusibl e ligan

d o r i s bound t o a non-diffusibl e complex . Thi s pape r i s a revie w

o f availabl e informatio nregardin g the subcellu lar distributio no f lead , effect s o ncellula r organelle s an d th e relationshi p o f thes e effect s t o symptom s o f lea d toxicity , an

d i s modifie d fro

m a nearlie r workin g pape r submitte d t o a tas k grou p onmeta l accumulation s (Goyer , 1972) .

SUBCELLULARLOCALIZATIO N O F LEAD

A numbe r o f technique s hav e bee nemploye d for measurin g the subcellula r distributio no f lead . Uptak e o f radioactiv e lea d iso tope s a t differen t tim e Interval s followin g intravenou s administra tionprovide s a dynami c mode l whereb y animal s ca nb e sacrified , an d the subcellula r distributio no f labelle d lea d measure d i n cellula r compartment s followin g separatio nb y differentia l centri fugation . Castellin o an d Alo j (1969 ) determine d th e distributio n o f lea d i nnuclei , mitochondria , microsome s an d cel l sa p o f live r an d kidne y

o f rat s for 9 day s afte r the intravenou s injectio no f Lea d entere d th e cell s withi non e hou r afte r injection . Inth e live r withi nth e firs t 2 4 t o 7 2 hour s ther e wa s a decreas e i nradioactivit y

i nth e nuclea r fractio nand a nincreas e i nmito chondria ; microsome s an d solubl e fraction s remaine d stable . Thes e

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relativ e distribution s remaine d constan t fo r nin e days . O nthe othe r hand , i nthe kidne y relativ e increase s o f lea d i nbot hnu clea r an

d mitochondria l fraction s occurre d durin g the firs t 2 4 hour s wit ha decreas e i nmicrosome s an d littl e chang e i nthe sol uble fraction . Afte r 2 4 hour s th e distributio nreache d a steady state . Mitochondria l bindin g o f lea d wa s remarkabl y

stron g an

d resiste d many

washings . Non e o f the rat s had bee npre-expose d t o excessiv e amount s o f lead .

i

- A simila r experimen t wa s reporte d b y Ba r It r or

i an d coworker s (1971) . The y

measure d the subcellula r distributio no f lea d i n heart , liver , kidne y an d splee nfollowin g intraperitonea l admini stratio no f 203pb . cell s wer e fractionate d int o nuclear , mitochon drial-lysosomal , microsoma l an d solubl e fraction s betwee n2 hour s an d 1 4 day s afte r administratio no f the isotope . Rena l tissu e wa s furthe r separate d Int o lysosoma l an d mitochondria l fractions ; mos t o f the lead wa s i nth e mitochondria l fraction . However , pre treatmen t o f the rat s wit hlea d diminishe d the in-vlv o uptak e o f radioactiv e lea

d b y the mitochondri a suggestin g tha t lead bindin g site s o nth e mitochondrio nma y b e saturated . However , isolate d ra t live r mitochondri a incubate d wit hlea d for m lead-containin g granule s i nthe inne r compartmen t (Walton , 1973) . Formatio no f thes e granule s require s the presenc e o f substrat e an d AT P an

d i s reduce d i nth e presenc e o f metaboli c inhibitor s suc ha s actlno myci nA o r dinitropheno l suggestin g tha t lea d accumulatio ni nmito chondri a i s a nactive , energy-dependen t process . Direc t measuremen t o f lea d conten t o f subcellula r component s o f the ra t kidne y

ha s show

ntha t the majo r fractio no f the lea d i s presen t i nthe nucleu s o f bot hcontro l an d lea d toxi c rat s bu t durin g excessiv e exposur e t o lea d b y fa r th e greates t incremen t i s i nthe nucleu s (Goye r e t al. , 1970b) .

NUCLEARINCLUSION

BODIE S

A characteristi c cellula r reactio ni nlea d Intoxicatio ni s the formatio no f discrete , dens e staining , intranuclea r Inclusio n bodies . Thes e wer e initiall y observe d b

y Blackma n(1936 ) some 3 7 year s ag o i nhepati c parenchyma l cell s an d rena l tubula r linin g cell s o f childre ndyin g o f acut e lea d encephalopathy . Mor e recentl y the y hav e bee nreporte d i nliver s an d rena l tubula r lining cell s o f •win e (Watrac han

d Vatter , 1962) , dog s (Stow e e t al , 1973) , rabbit s (Has s e t al. , 1964) , fowl s (Simpso ne t al. , 1970 ) an d l nkidney s onl y o f rat s (Goyer , 1968) . Th e ultrastruetur e o f a typica l inclusio nbod y i nthe nucleu s o f a rena l tubula r linin g cel l o f a lead-Intoxicate d ra t consist s o f a dens e centra l cor e an d a noute r fibrillar y zone . The y

ar e alway s independen t o f nucleoli . Inclusio nbodie s var y considerabl y

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iOYE R AN D J

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. MOOR E

' days . O

n th e ea d i n bot h nu th e firs t2 4 ang e i n th e sol eache d astead y 'bl y stron g an d "\ pre-expose d t o

o an d coworker s o n o f lea d i n rltonea l admini
nuclear , mitochon
betwee n 2 hour s Rena l tissu e wa s

• 1 fractions ; n . However , pre - -viv o uptak e o f na tlea d bindin g ever , Isolate d ead-contalnln g Formatio n o f an d AT Pan d i s :uc h a s actlno umulatlo n l n mlto -

.lula rcomponent s o n o f th e lea d i s oxi c rat s bu t ices tincremen ti s

ntoxicatio n i s .uclea rinclusio n

. n (1936 ) som e 3 7 tubula rlinin g •chy . Mor

e recentl y a rlinin g cell s o f al , 1973) , rabbit s an d i n kidney s

>d y i n th e nucleu s te d ra tconsist s one . The y ar e var y considerabl y CELLULAR EFFECT S O F LEA D 449
i n siz e an d i n rati o o f cor e t o oute rfibril s (Bracke n e tal. , 1958 ; Beaver , 1961 ; Richte re tal. , 1968) . Richte re tal . (1968 ) estimat e th e thicknes s o f th e fibril s t o rang e fro m 10 0 £ t o 13 0 £ , althoug h th e siz e o f fibril s varie s wit h fixatio n an d stainin g pro cedures . A fin e structur e t o th e fibril s (periodicity ) ha s no t bee n observed . Th e compositio n o f lead-induce d intranuclea rinclu
sio n bodie s ha s bee n furthe rstudie d b y histochemistry , autoradio
graph y an d b y direc tanalysi s afte risolation . Histochemica l studie s hav e bee n summarize d b y Richte re tal . (1968) . Inclusio n bodie s d o no tstai n wit h th e Feulge n reaction , althoug h the y ar e sometime s surrounde d b y Feulgen-positlv e material ; The y d o no c stai n wit h fas tgree n afte rtreatmen twit h trichloroaceti c acid , bu td o stai n strongl y wit h mercuria l bromopheno l blu e an d b y basi c fuchsi n suggestin g the y contai n protein , bu tprobabl y no thistones . The basi s o f

th e acid-fas treactio n i s uncertain ; i tma y simpl y b e aphysica l phenomenon . Landin g an d Naka l (1959 ) showe d tha tth e inclusion s contai n a tmos tsmal l amount s o f lipi d an d suggeste d th e acid-fas tpropertie s o f thes e bodie s wer e relate d t o th e pre senc e o f

sulfhydry l group s i n th e protein . Th e presenc e o f

lea d i n th e inclusio n bodie s wa s suspecte d bu tunprove d fo rman y years . Finne ran d Calver y (1939 ) staine d section s o f

kidney s fro m lea d poisone d rat s wit h hydroge n sulfid e an d foun d particle s o f lea d sulfid e occurrin g I n nucle i o f tubula r epitheli apresumabl y correspondin g t o intranuclea rinclusio n bodies . Othe rattempt s t o identif y th e meta l eithe rhistochemlcall y o rb y x-ra y diffractio n hav e bee n unsuccessfu l (Watrac h an d Vatter , 1962) . Mor e recently , lea d withi n th e intranuclea rInclusio n bod y ha s bee n positivel y demonstrate d b y autoradiograph y (Dallenbach , 1965 ) an d electron-prob e x-ra y microanalysi s (Carrol l e tal. , 1970) . Th e structur e an d compositio n o f th e inclusio n bodie s hav e bee n furthe rstudie d afte risolatio n b y differentia l centrifugatio n and sonicatio n (Goye re tal. , 1970b) . Treatmen to f Inclusion s i n tissu e section s (Richte re tal. , 1968 ) o rIsolate d inclusio n bodie s (Goye re tal. , 1970b ) wit h proteolyti c enzyme s result s i n partia l digestion , bu tthe y ar e no taltere d b y incubatio n wit h DNAase o r RNAase . Direc tchemica l analysi s o f Isolate d inclusio n bodie s con firm s tha tthe y ar e compose d o f alead-protei n comple x containin g approximatel y 5 0 u g o f lea d pe rm

g protein . Lea d withi n th e inclu
sio n bodie s i s 6 0 t o 10 0 time s mor e concentrate d tha n whol e kidne y lea d (Goye re tal. , 1970b ; Horn , 1970) . Amin o aci d compositio n an d solubilit y characteristic s o f th e Inclusio n bod y protei n resembl e thos e o f

th e residua l acidi c fractio n o f protein s i n norma l nucle i (Goye re tal. , 1971) . Th e protei n ha s hig h conten to f asparti c and glutami c acids , glycine , cystine , an d tryptophan . The inclusio n bodie s ar e insol
ubl e l n physiologica l medi abu tma y b e dissolve d i n denaturant s



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. GOYE R AN D J

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. MOOR E

lik e 6 M

ure a an

d sodiu m deoxycholate . Th e lea d i s removabl e b

y incubatio nin-vitr o wit hchelator s lik e EDTA . I nthi s circumstanc e th e inclusio nbody

lose s it s typica l morphologica l structur e an d become s a namorphou s protei nresidu e whic hcontinue s t o b e insol ubl e (Moor e e t al. , 1973) . Th e inclusion s als o contai nlesse r amount s o f othe r metal s includin g calcium , iron , zinc , coppe r an d cadmiu m i norde r o f decreasin g concentrations . A scheme ha s bee npropose d fo r the possibl e rol e o f th e intra nuclea r inclusio nbody I nlead poisonin g (Goyer , 1971) . I t l s suggeste d tha t th e inclusio nbody serve s a s a nadaptiv e o r protec tive mechanis m

durin g transcellula r transpor t o f lead . I nthe cours e o f excretio no f lead fro m capillar y

t o bil e l nhepati c cells , o r b

y transtubula r flo

w i nrena l tubula r linin g cells , a por tiono f the lead enter s the nucleu s wher e I t become s bound int o a lead-protei ncomple xan d l s n o longe r diffusible . Thi s mechanis m ha s th e effec t o f maintainin g a relativel y lo w cytoplasmi c concen tratio no f lead and , therefore , reducin g th e toxi c effect s o f lea d o nsensitiv e cellula r functions , suc ha s mitochondria l respiratio n an d protei nsynthesis . Indirec t suppor t fo r thi s hypothesi s i s foun d i nexperimenta l studie s o f tb e relationshi p o f the formatio no f intranuclea r inclu sio nbodie s an

d othe r rena l effect s o f lead . Whe ngroup s o f rat s ar e give ndifferen t concentration s o f lead i nthei r drinkin g water , intranuclea r inclusio nbodie s ar e observe d a t a lowe r dos e thani s any othe r rena l effec t o f lead ; i nfact , a t a lowe r dos e o f lead thantha t whic hproduce d sign s an d symptom s o f lead toxicity . In clusio nbodie s appea r t o b e relate d t o rena l lea d conten t an

d dis cernibl e i nthe ra t model s whe nkidne y lea d concentratio ni s be twee n1 0 an

d 2 0 ug/ g we t weight . Th e lead-induce d nuclea r Inclusio nbody ma y als o facilitat e urinar y excretio no f lead . The y ar e foun

d i nth e urin e o f childre n wit hacut e lea

d poisonin g (Landin g an d Nakai , 1959) . Ina recen t study o f rena l biops y

materia l fro

m five lea

d industr y

worker s wit hvaryin g time s o f exposur e t o lea d fro m 1 6 week s t o mor e than 2 0 years , typica l lead-induce d inclusion s wer e onl y foun d i nnucle i o f worker s employe d for les s tha non e yea r (Crame r e t al. , i npress) . Urinar y excretio no f lea

d wa s highe r l nthos e worker s wit hinclusio n bodie s tha nl nolde r worker s withou t suc hcomplexe s althoug hothe r symptom s o f lea

d toxicit y

wer e mor e marke d l nth e latte r group . Whethe r the absenc e o f Inclusio nbodie s i nthes e workme nreflect s repeate d chelatio ntherap y o r implie s a nalternativ e cellula r meta bolis m o f lea d i s no t known . A simila r study

o f rena l biopsie s fro m 2 3 lead industr y

worker s wit hvaryin g period s o f exposur e t o lea d ha s bee nreporte d by Riche t and coworker s (1964 , 1966) . Althoug hsome for m o f a nuclea r body wa s foun

d i n1 7 o f the 2 3 rena l biopsies , onl y on e showe d the

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i d I s removabl e b

y I nthi s circumstanc e ca l structur e an

d

. inue s t o b e insol contai nlesse r

, zinc , coppe r and

2 rol e o f the intra -

. 1971) . I t I s idaptiv e o r protec 5 lead . I nth e li e i nhepati c lining cells, a por ose s bound int o a Thi s mechanis m ytoplasmi c concen i c effect s o f lea

d ndria l respiratio n

n d l nexperimenta l intranuclea r inclu a ngroup s o f rat s 2i r drinkin g water , lowe r dos e thani s /e r dos e o f lead ;a d toxicity . In - ' conten t an

d dla ntratio ni s be als o facilitat e urin e o f childre n 9 ) . I na recen t jstry worker s ek s t o mor e tha n ly foun d i nnucle i

- e t al. , i npress) , :er s wit hInclusio n > s althoug hothe r latte r group , orkme nreflect s I'v e cellula r meta -

• d industr y

worker s reporte d b y

Riche t a nuclea r bod y 

### showed th e CELLULA R EFFECT S O F LEA D 

### 45 1 typica l morpholog y

o f th e characteristi c lead-induce d Inclusio n body an d thi s wa s the onl y individua l i nthe grou p wit ha histor y o f shor t ( 3 months ) exposur e t o lead . Othe r nuclea r bodie s occur ring i nlea

d toxi c worker s resemble d granula r chromati nmasse s whic hoccu r i na numbe r o f pathologica l condition s characterize d by cellula r hyperactivit y (Bouteill e e t al. , 1967) . Inclusio nbodie s occu r i nnucle i o f osteoclast s o f lea d poi sone d pig s (Hs u e t al. , 1973) . Thei r formatio ni s augmente d b y lowerin g dietar y

calcium,suggestin g tha t lea d i s reabsorbe d durin g osteolysi s similarl y t o calcium . Lead becomea'boun d int o th e non dlffuslbl e inclusio nbody . Th e lead-protei ninclusio nbody phenomeno ni s no t restricte d t o anima l cel l nucle i bu t als o occur s i nplan t cells . Bryophyte s (moss ) ar e know

nt o accumulat e heav y metal s i nconcentration s whic har e toxi c fo r othe r group s o f plants . I t ha s recentl y bee n show ntha t mos s cultivate d i na greenhous e and watere d dall y wit h lea d acetat e solution s form s lea d containin g inclusio nbodie s i n nucle i o f lea f cell s (Skaa r e t al. , 1973) . Simila r inclusio n bodie s ma y

occu r i nroo t cell s o f cor ngrow ni nsoi l wit hadde d lea d (persona l communication , Dr . D . E . Koeppe , Universit y o f Illi nois , Urbana , Illinois) .

EFFECT S O F LEA D O N

CEL L DIVISIO N

AN D PROTEI N

SYNTHESI S Althoug hlead ma y

b e concentrate d i nnucle i i nthe for m o f a protei ncomplex , the metaboli c effec t o f lea d localizatio no nnu clea r function , particularl y cellula r division , l s uncertain . Th e findin g o f granula r chromati nbodie s i nbiopsie s o f lea d worker s suggest s Increase d cel l turnove r time . Recen t studie s (Choi e an d Richter , 1972a ) hav e show ntha t a singl e Intraperitonea l dos e o f lea d wil l greatl y

enhanc e DN

A synthesi s an

d proliferatio no f rena l tubula r cell s withi n2 day s an d repeate d injection s give nl n2-day interval s wil l stimulat e successiv e wave s o f nuclea r DN A synthesi s i ntubula r epithelia l cell s (Choi e an d Richter , 1972b) . Thi s findin g i s o f particula r interes t sinc e lea d doe s hav e a carcinogeni c effec t i nrat s an

d mic e afte r long-ter m Intoxication . O nthe othe r hand , a carcinogeni c effec t fo r lead ha s no t bee nobserve d l nma n o r othe r specie s o f animals . Excessiv e exposur e t o lead doe s produc e structura l alteration s i nth e chromosome s o f ma nand experimenta l animals . Nuclea r poly ploidy an d abnormalitie s i nmitosi s hav e bee nnote d i nbon e marro w cell s b y a numbe r o f author s (Waldron , 1966 ) bu t chromosomal aber ration s i nlead Intoxicatio nhav e onl y recentl y

bee nrecognized . Chromosome s fro

m leukocyt e culture s fro m mic e fe

d a die t containin g I Z lead acetat e show a nincrease d numbe r o f gap-brea k typ e aberra tion s usuall y

involvin g onl y

singl e chromatid s (Mur o an d Goyer ,

'•Z&AirZrri&J?-'-£mf!!• ! • 

### 45 2 R . A . GOYE RAN D J . F . MOOR E 1969) . Simila r gap-brea k type s wer e als o foun d l nlea

d worker s wit hbloo d lea

d level s rangin g fro m 6 2 t o 8 8 ug/10 0 g (Schwantlt z e t al. , 1970) . I naddition , a variet y o f non-specifi c change s i n chromosome morpholog y wa s als o presen t suc ha s adhesion s an d spl ralizin g defects . Tetraplol d mitosi s an d the mitoti c inde xwer e als o Increase d i nthe workers . Moreover , th e percentag e o f abnor ma l mitose s correlate d ver y wel l wit hurinar y

d-ALA excretion . Simila r studie s hav e no t bee nperforme d o nacutel y intoxicate d childre nno r i s th e mechanis m for thi s lead effec t known . Lead , lik e othe r heav

y metals , doe s hav e a naffinit y for nuclei c aci d bindin g bu t whethe r lead become s biochemicall y boun d to-chromo some s it » viv o l s no t known . Th e effec t o f lead bindin g t o nuclea r protein s o ncel l divi sio ni s als o Incompletel y understood . Lea d i s bound t o insolubl e nuclea r protein s a s discusse d I n the formatio no f nuclea r inclusio nbodies . Lead i s als o associate d wit hNaO H solubl e acidi c proteins . Acidi c nuclea r protein s ar e Implicate d i nthe regulatio no f DN A replicatio nan

d influenc e DNA dependen t RN

A synthesi s (Stei nand Baserga , 1972) . Ther e l s some Informatio nt o sugges t tha t lea d ma y affec t protei nsynthesi s a t the leve l o f the ribosome . Waxma nan d Rablno vit z (1966 ) and Ulme r an d Valle e (1969 ) observe d tha t lea d cause s disaggregatio no f polyribosomes . Th e latte r worker s als o reporte d inhibitio nb

y lead o f leucin e incorporatio nInt o t-RN A i nE . coli . Purifie d RN

A i s depolymerize d b y lea d (Farkas , 1968) .

EFFECT S O F LEAD ON

MITOCHONDRI A Th e affinit y

an d relativel y

stron g bindin g o f lead t o mito chondria l membrane s suggest s tha t thi s organell e i s a particularl y susceptibl e targe t o f the subcellula r effect s o f lead . Lea d toxicit y

i nhuman s an

d experimenta l animal s i s associate d wit halteration s i nultrastruetur e an d functio no f mitochondri a i n hepati c parenchyma l cells , rena l proxima l tubula r lining cell s an d reticulocyte s i nthe bon e marrow . Th e principl e clinica l manifes tation s o f lea

d toxicit y

involv e thes e organ s a s wel l a s the centra l nervou s system . Althoug hdisturbance s i nmitochondria l structur e an d functio nar e no t the onl y subcellula r effect s o f lead , i t i s suggeste d tha t the tnviv o effec t o f lead o nthes e organelle s con tribute s t o th e anemi a o f lead poisonin g an d impaire d rena l tubula r function . Th e relationship o f th e mitochondria l effect s o f lea d t o othe r symptom s o f lead toxicit y ar e les s wel l understood . Mitochondri a i nproxima l .rena l tubula r linin g cell s o f chil dren , adult s and experimenta l animal s wit hlea d poisonin g show

«j%»*a»ia6**4S»:«»a»K ;\. GOYE R AN D J

. F

. MOOR E 

### i n lea d worker s '10 0 g (Schwantit z •ecifi c change s i n dhe8lon s an

d spi
- 'toti c inde x were rcentag e o f

abnor AL A excretion . l y intoxicate d c tknown . Lead , o r nuclei c aci d

' ound t o chromo in s oncel l divi a s discusse d l n I s als o associate d r procein s ar e n d influenc e DNA a d ma y affec t 'axma nan d Rablno tha t lea

d cause s er s als o reporte d t-RN A I nE . coli . •58) .

lead t o mito I I s a particularl y 

### I '-ead . -al s I s associate d c mitochondri a i n lining cell s an

d linlca l manlfes el l a s the central Jdria l structur e |o f lead , i t l s organelle s con Ira d rena l tubula r Iffect s o f lea

d fnderstood . cell s o f chil lisonin g show CELLULAR EFFECT S O F LEA D 

### 45 3 varyin g degree s o f swellin g wit hmarginatio no f crista e (Angevin e e t al. , 1962 ; Cohe ne t al. , 1960 ; and Goyer , 1968 ) probabl y secon dar y t o increase d membran e permeability . Suc hchange s ar e simila r t o the non-specifi c swellin g tha t occur s i nth e earl y stage s o f othe r form s o f cellula r Injur y an d i s probabl y

reversible . Th e fat e o f suc hmitochondri a i s uncertai nbu t increase d number s o f myeli nfigure s and autophagocytosi s sugges t a nIncrease d rat e o f turnover . Mitochondri a l ncell s o f rena l biops y specimen s fro

m lea d industr y

worker s show

evidenc e o f buddin g an d division . Lea d induce d granule s o r Inclusio nbodie s simila r t o thos e forme d i n nucle i ma y

b e produce d .I

nvitr o i nisolate d live r mitochondri a (Walton , 1973 ) bu t thes e hav e no t bee nnote d i nvivo . Othe r type s o f intramitochondria l inclusion s hav e bee nob serve d bu t ar e probabl y no t specifi c fo r lead poisoning . Watrac h (1964 ) ha s describe d a nintramitochondria l lamella r typ e crystal loi d i nlive r parenchyma l cell s o f lea d poisone d swine . Thes e ar e compose d o f a nintersectin g band o f fine , loosel y packe d array s tha t runparalle l and perpendicula r t o for m a lattic e pattern . Suc hlamella r formation s ar e characteristi c o f chroni c cellula r injur y accompanyin g a numbe r o f metaboli c abnormalitie s suc ha s diabete s mellitus , alcoho l intoxicatio nand biliar y obstructio n a s wel l a s experimentall y induce d hypoxi a (Shirak i an d Neustein , 1971) . From th e observe d alteration s i nmitochondria l ultrastructur c some impairmen t o f functiona l abilit y i s suggested . Nevertheless , mitochondri a fro

m cell s o f subject s wit hchroni c intoxicatio nar e expecte d t o hav e som e degre e o f respirator y abilit y

sinc e cellula r necrosi s o r irreversibl e cellula r injur y i s no t a prominen t morpho logi c feature . A

decrease d leve l o f mitochondria l functio ni s com patibl e wit hth e concep t o f a naltere d functiona l steady stat e o r lowere d leve l o f cellula r activity . Measuremen t o f respirator y activit y

o f mitochondri a fro m live r o r kidne y

o f lead-treate d experimenta l animal s confirm s thi s con cept . Tera s and Kakh n(1967 ) foun d decrease d respirator y rate s in . mitochondri a o f rabbit s treate d wit hdail y intraperitonea l injec tion s o f lea

d acetat e (1 0 t o 2 0 mg/k g body weight ) fo r seve nmonths . AD P stimulate d respiratio nwa s lowe r fo r mitochondri a fro m lead treate d rabbit s i nth e presence o f thre e differen t substrates : a ketoglutarate , pyruvate and succinate . Phosphorylatio nwa s als o decreased . Th e author s sugges t respirator y impairmen t i s mos t pro nounce d usin g pyruvat e a s substrate . Studie s wit hmitochondri a isolate d fro m kidney s o f lead poi sone d rat s show

a specifi c Impairmen t i nrespirator y and phosphory lativ e abilitie s i npyruvate-dependen t respiratio n%s evidence d b y altere d respirator y control (RCR ) and ADP: 0 ratios . Slightl y highe r Stat e I

V an d Stat e II I rate s o f toxi c mitochondri a I nth e

# i '



# 1 > 45 4 R

. A

. GOYE R AN D J

. F

. MOOR E



## I i 

# I * presenc e o f pyruvat e sugges tpartia l uncouplin g o f phosphorylation . Thes e functiona l parameter s whic h ar e reflecte d b y RC Ran d ADP: 0 ar e no tdifferen tfro m th e contro l mitochondri ai n succinate-depen
den trespiratio n (Goye ran d Krall , 1969a) . Sinc e succinat e oxida
tio n an d relate d electro n transpor tby-pas s th e pyruvate-NA D reduc tas e enzym e complex , i tI s suggeste d tha tth e NA D reductas e comple x i s mor e sensitiv e t o lead . A mor e recen trepor tindicate s tha t magnesiu m restore s oxidativ e abilit y an d dinitropheno l sensitivit y I n lea d toxi c mitochondri afo rNA D couple d respiration , bu tI tha s n o effec to n succinate-supporte d respiratio n (Kral l an d Dougherty , 1971) . Decrease d oxyge n uptak e rat e fo rbot h Stat e II I an d I V i n succinate-supporte d respiratio n i n lea d toxi c mitochondri ama y b e evidenc e o f

decrease d succinoxldas e enzym e whic h correlate s wit h decrease d mitochondria l protei n (Rhyn e an d Goyer , 1971) . Ulme ran d Valle e (1969 ) conclud e tha tlea d act s specificall y wit h dithio l bond s i n th e llpoamld e dehydrogenas e l n th e pyruvat e dehydrogenas e systen tbu tthi s wa s validate d b y l n vitr o workonly . Thi s sit e l s sensitiv e t o othe rmetal s suc h a s mercur y an d cadmium bu tl tI s uncertai n whethe rthi s sit e i s accessibl e t o lea d i n th e i n viv o system . Th e effec to f

lea d o n mitochondri ao f bon e marro w reticulo
- cyte s i s o f

particula rinteres tbecaus e o f th e resultan timpairmen t i n heme synthesi s and subsequen tclinica l anemia . I tals o serve s a s afairl y wel l define d exampl e o f atoxi c chang e l n a n Intramito
chondria l enzym e system . A tleas tthre e step s l n heme synthesi s ma y b e affecte d b y lead . Inhibitio n o f d-aminolevullni c aci d dehydratas e (AIA-D ) i s probabl y th e enzym e i n th e hem e pathwa y tha ti s mos tsensitiv e t o lead . Inhibitio n o f thi s enzym e result s i n abloc ki n utilizatio n o f d-aminolevullni c aci d (d-ALA ) an d subsequen tdeclin e i n heme synthesis . Unde rth e propose d schem e o f negativ e feed-bac kcontro l o f heme synthesi s a s propose d b y Granic kan d Lever e (1964) , d-AL A synthetas e activit y i s derepresse d resultin g I n increase d activit y an d synthesi s o f

d-ALA . Thi s ma

y b e regarde d a s asecon d effec t o f lea d o n hem e synthesis . Sinc e utilizatio n o f d-AL A i s blocked , urinar y excretio n i s increased . Assa y o f urinar y d-AL

A serve s a s asensitiv e inde x o f abiochemica l lea d effec tan d i s widel y use d a s a n indicatio n o f lea d toxicit y (Haeger-Aronsen , 1960) . A thir d abnormalit y

i nhem e synthesi s describe d i nlea d in toxicatio ni s inhibitio no f th e enzyme , ferrochelatas e (hem e syn thetase ) (Otrzonsek , 1967 ) whic hi s locate d onthe inne r membran e o f mitochondri a (Jone s an d Jones , 1968) . Thi s effec t I s particu larl y Interestin g becaus e o f associate d ultrastructura l change s l nth e mitochondrion . Ferrochelatas e catalyze s the incorporatio n o f th e ferrou s lonint o the porphyri nrin g structure . Bessl s an d Jense n(1965 ) hav e show ntha t ironI nth e for m o f apoferritl nan

dP.1 0 

### •.jsasEsHa&stt. ' ::ij"LrnirFii'i :

'E R AN D J

. F.MOOR E ohosphorylation . IC Ran d ADP: 0 <:uccinate-depen
- -cinat e oxida
rvate-NA D reduc eductas e comple x

• I icate s tha t | *^o l sensitivit y •on , bu ti tha s an d Dougherty , II I an d I

V i n 'loadrl ama

y b e |"relate s wit h '71) . specificall y l" » th e pyruvat e tr o wor konly . •r y an d cadmium t o lea d l n th e

•o w reticulo
- tan tImpairmen t I tals o serve s

• n a n Intramito
ffe e te d b y 's e (ALA-D ) l s sensitiv e t o |l nutilizatio n iin e i nhem e Ied-bac kcontro l (1964) , d-AL

A ease d activit y econ d effec t L A l s blocked , AL A serve s a s s widel y use d 960) . i n lea d in
s e (hem e syn nne rmembrane i s partlcu ra l change s Incorporatio n Bessi s an d ?ferriti n an d CELLULAR EFFECT S O F LEA D 

### 45 5 ferruginou s micelle s accumulate s i nmitochondri a o f bon e marro w reticulocyte s fro

m lea d poisone d rats . Gibso nan

d Goldber g (1970 ) recentl y reporte d n o Inhibitio no f thi s enzyme (ferrochelatase ) i nhomogenate s prepare d fro m live r o f lea d toxi c rabbits , bu t whe nthe y adde d lea

d acetat e t o th e homo genat e l

nconcentration s simila r t o thos e presen t i nthe origina l unhomogenize d tissues , a marke d inhibitio noccurred . Lead asso ciate d wit hth e enzyme i nviv o ma y b e s o loosel y

bound a s t o b e los t i nth e isolatio nprocedure . Nevertheless , the experimen t doe s show

tha t ferrochelatas e i s readil y inhibite d b

y lead . Othe r step s i nth e biosyntheti c pathwa y o f hem e ma y

als o b e abnorma l i nlea

d toxicit y

bu t th e evidenc e fo r thes e i s incomplete . Increas e i nurinar y excretio no f coproporphyrin , th e degradativ e produc t o f coproporphyrinoge nIII , i s a sensitiv e reflectio no f lea d toxicity . Metabolis m o f porphobilinoge nt o coproporphyrinoge n proceed s unimpaired . However , sinc e ALA- D i s a nextra-mitochondrIa l enzyme , an

d late r step s i nhem e synthesi s ar e intramitochondrial , coproporphyrinogenmus t re-ente r th e mitochondrio nt o b e metabo lized further . I t ha s bee nsuggeste d tha t transpor t o f metabolite s lik e coproporphyrinoge nint o th e mitochondria l matri xmigh t b e im paire d b

y altere d inne r membran e permeability , o r decrease d effi cienc y i noxidatio nan

d phosphorylatio n(Haeger-Aronse ne t al. , 1968) . Whethe r th e increase d urinar y coproporphyrinoge noccurrin g I nlead poisonin g l s a reflectio no f a non-specifi c alteratio ni n mitochondria l membran e o r reflect s a mor e specifi c effec t o f lea d o nth e intramitochondria l enzyme coproporphyrinogenase , i s no t known . Th e principa l clinica l manifestation s o f lead toxicit y Involv e thre e orga nsystems : the centra l nervou s system , hematopoieti c system an d the kidney . A

relationshi p betwee nth e mitochondria l effec t o f lea

d and th e centra l nervou s system patholog y

ha s no t bee ndemonstrated . However , functiona l impairmen t o f mitochondri a o f reticulocyte s i s responsible , a t leas t i npart , fo r th e anemi a o f lea d poisoning , an

d lead toxi c mitochondri a o f reticulocyte s ar e responsible , a t leas t i npart , for the anemi a o f lead poisoning , an d lea d toxi c mitochondri a i nproxima l rena l tubula r linin g cell s mus t decreas e th e functiona l abilit y o f thes e cells . Transpor t o f a numbe r o f substance s includin g sodium , potassium , phosphat e ion s an d smal l organi c molecule s suc ha s amin o acid s an d glucos e occur s i nthi s portio no f th e rena l tubul e an d i s a nactiv e proces s dependen t onAT P largel y derive d fro

m mitochondria l oxidativ e phos phorylation .
I nacut e lea

d poisoning , particularl y i nchildren , excessiv e excretio no f amin o acids , glucos e and phosphat e occur s (Chisolm , 1962) . Whethe r reduce d reabsorptio no f thes e substance s i nlea d i r

»'. i .'. i

i 4 1

» . " ': 1

-jW*^^*" 8 * ^ ^ j»<^»£aa*^i*\\«Kss>*-»=»T- -Mi%e£>&k&mJk.



### 45 6 R . A . GOYE RAN D J . F . MOOR E poisonin g i s entirel y relate dt o reduce denerg y productio nb

y lea dtoxi c mitochondri a o r i s als o a consequenc e o f a lea deffec t o nothe r cellula r membrane s i s uncertain . Mos t o f the energ y syn thesize dl nproxima l tubula r linin g cell s i s require dfor sodiu m transport . I t i s interestin g tha t Sandstea de t al . (1970 ) hav e recentl y

show ntha t me nwit hchroni c lea dpoisoning , followin g excessiv e industria l exposur e t o lea do r ingestio no f lead-contami nate dIllici t moonshine , d o hav e reduce dsodiu m reabsorpclon . Thi s defect , lik e th e aminoaciduria , i s reversibl e b y treatmen t o f th e . lea dpoisonin g wit hchelatin g agent s (EDTA) .

r Respirator y

rate s o f bon e marro w reticulocyte s fro

m lea dintoxi cate drat s ar e les s tha nnorma l (Lessle r e t al. , 1968) , probabl y reflectin g impaire dmitochondria l respiration . Als o some o f th e enzyme s associate dwit hheme synthesi s ar e locate deithe r withi n th e inne r mitochondria l membran e o f matri xo r ar e i nclos e associa tio nwit hthi s organell e an d ar e dependen t o nintac t mitochondria l respiration . Some impairmen t o f heme enzyme functio nma y follo w th e leadeffec t o nrespiration . Th e effec t o f lea

do ncellula r conten t o f othe r hem e protein s suc ha s cytochromes , catalas e an d peroxidase s ha s receive dlimite d study . Prade r an

d Vannott i I

na nattemp t t o compar e effect s o f leadpoisonin g o nhemoglobi nan d othe r cellula r porphyri nmoietie s measure dlevel s o f hemoglobi nan d cytochrom e c i nrabbi t liver . In contras t t o decreas e i nth e hemoglobi nvalue s cytochrom e c leve l remaine dconstan t (Vannotti , 1955) . However , measuremen t o f the tota l cytochrom e conten t o f ra t kidne y mitochondri a b

y differenc e spectr a showe da selectiv e decreas e l ncytochrom e 88 3 i nlea dpoi sonin g (Rhyn e an

d Goyer , 1971) . Whethe r thi s decreas e resulte dfro m inhibitio no f mitochondria l protei nsynthesi s whic hi s reflecte d b y decreas e I ncytochrom e 88 3 o r specifi c inhibitio no f th e cyto chrom e 88 3 heme moiet y ha s no t bee nresolved . Th e protei nmoiet y a s wel l a s th e hem e moiet y differ s fo r eac hclas s o f cytochrome s s o inhibitio no f eithe r fractio nwoul dresul t i na decreas e o f th e cytochrome .

LEAD AN D LYSOSOME S Th e radioisotop e studie s o f Barltro pan d coworker s (1971 ) sugges t tha t lea

dha s les s affinit y fo r iysosome s tha nothe r sub cellula r organelles , particularl y mitochondri a an

d nuclei ; no r doe s leadintoxicatio ni nth e ra t provok e an

y remarkabl e increas e i nlysosoma l enzyme s (Krigma ne t al. , 1968) . Th e origi no f the lead-induce dincreas e i nurinar y ^-glucuronidas e note db y thes e worker s i s uncertai nbu t ma y b e derive dfro

m a lea deffec t o nendo plasmi c reticulu

m (Krigma ne t al. , 1970) . Autophagocytosi s o f lea d containin g mitochondri a ma y resul t inlea

dcontainin g secondar y Iysosome s a s suggeste dfro m observatio no f biops y specimen s fro

m



# p i

^i^WS^:fe^ ; H»>*^ isaW r.i r=UJllfcrrs:, ! ;_i23mTrjE££?y ••jMmm&^Sit *fi ' 

# zm^mmi P.M

50YE R AN D J

. F

. MOOR E CELLULAR EFFECT S O F LEA D 45 7 >roductio n b y ' f alea d effec t th e energ y

syn re dfo r sodiu

m * • (1970 ) hav e g , followin g o f lead-contami absorpcion . Thi s reatmen t o f th e

s fro m lea dintoxi 968) , probabl y 's o some o f the eithe r withi n i nclos e associa -

~ t mitochondria l '.o nma y follow

•» r hem e protein s -eceive dlimite d

- e effect s o f phyri nmoietie s -abbl t liver . I n hrome c leve l remen t o f th e b y differenc e -8 3 i nleadpol as e resulte dfro

m • i s reflecte d no f th e cyto protei nmoiet y f cytochrome s decreas e o f the

Uer s (1971 ) n a nothe r sub liuclei ; no r |<abl e increas e igi no f th e |->te d b y thes e •ffec t o nendo - |>cytosi s o f lead i g secondar y hecimen s fro

m lea dworker s (Crame r e t al. , i npress) . Also , lea dha s bee n identifie db

y x-ra y microanalysi s i ndens e cytoplasmi c concretion s i nkidne y

cell s o f rat s administere dlarg e dose s o f lea dintraperi toneall y

(Murakami , 1971) . Suc hconcretion s hav e no t bee nfoun d followin g oral administratio no f lea di nrat s an

d l nhuma nbiopsy 9 material .

SUMMARYAN D CONCLUSION S 1 • f Th e intracellula r localizatio no f lea dha s bee nbes t studie d i nlive r an

d kidney , th

e tw o organ s mos t concerne dwit htranscellu lar transpor t an

d excretio no f lead . Withi nthe cel l lea dha s the greates t affinit y

fo r mitochondria l membrane s an d nuclei . Nuclea r lea dbind s wit hanacidi c protei nfractio nan d accumulate s t o for m a discret e inclusio nbody whic hha s a characteristi c ultrastrueture . Whe nthe rena l tubula r cell i s presente dwit heve nlarge r concen tration s o f lea

dintracytoplasmi c concretion s ar e formed . Th e relationshi po f thes e concretion s t o th e lysosoma l system ha s no t bee ndemonstrate dbu t dens e stainin g bodie s d o for m l nsecondar y Iysosome s derive dfro m degeneratin g leadtoxi c mitochondria . Th e effec t o f leado nmembran e enzyme system s an d biochemica l activit y

o f subcellula r organelle s i s relate dt o the patter no f intracellula r partitioning . Th e lea deffec t o nmitochondri a de crease s th e abilit y o f cell s t o for

m specifi c functions , e.g . trans por t function s i nproxima l rena l tubula r linin g cells . Sensitivit y o f enzyme s concerne dwit hheme synthesi s result s l nanemi a a s wel l a s increase durinar y excretio no f intermediat e product s o f th e hem e synthesizin g pathway . Measurabl e amount s o f lea dar e presen t i nmos t cell s o f person s l nth e genera l populatio nwithou t apparen t advers e effect . Wit ha n increas e inlea

dburde nthe transitio nfro m n o effec t t o toxicit y i s probabl y

no t sharpl y

definabl e bu t reflect s a spectru m o f cellula r events , some compensatory , som e pathological . Knowledg e o f the intracellula r distributio no f lea dan d it s effec t o nsubcellula r component s shoul dImprov e the abilit y t o discer nbetwee na tolerabl e an dtoxi c lea

dburden .

REFERENCE S Angevine , J.M. , Kappas , A. , Degowln , R.L. , Spargo , B.H. : Rena l tubula r nuclea r inclusion s l nleadpoisoning . A clinica l an

d experimenta l study . Arch . Patho L 73:486-494 , 1962 . Barltrop , D. , Barrett,'A.J. , Dingle , J.T . butlo no f lea

di nthe rat . J . Lab . Clin . Med . Subcellula r distri * 77:705-712 , 1971 .

• KtSthm X-j^m^iif-m^^i^^jjfc^gSfcMJ * ,»«mite

. A.-stm^*"!***'Hi: - P



### 45 8 R . A . GOYE RAN D J . F . MOOR E

Beaver , D.L. : Th e ultrastructur e o f th e kidne y i nlea dintoxi catio nwit hparticula r referenc e t o Intranuclea r inclusions . Am . J . Pathol . 39:195-208 , 1961 . Bessls , M.C , Jensen , W.N. : Sideroblasti c anaemia , mitochon dri a an d erythroblasti c iron . Br . J . Haematol . 11:49-51 , 1965 .

ml.,. Blackman , S.S. , Jr. : Intranuclea r inclusio nbodie s i nkidne y

an dlive r cause db y lea dpoisoning . Bull . John s Hopkin s Hospita l 58:384-403 , 1936 . 

# i Bouteille , M. , Kallfat , S.F. , Delareue , J .

variation s o f nuclea r bodie s l nhuma ndiseases . Res . 19:474-486 , 1967 . 

### Ultrastructura l J . Ultrastructura l

Bracken , E.C , Beaver , D.L. , Randall , C.C : Histochemica l studie s o f vira l an d lead-induce

d Intranuclea

r bodies . J . Path . Bact . (Lond ) 75:253-256 , 1958 . Carroll , K.G. , Spinelli , F.R. , Goyer , R.A. : Electro nprob e •icroanalyze r localizatio no f lea

d i n kidne y tissu e o f poisone

d rats . Natur e (Lond ) 227:1056 , 1970 . Castellino , N. , Aloj , S. : Intracellula r distributio

no f lea

d inth e live r an d kidne y o f th e rat . Br . J . Ind . Med . 26:139 - 143 , 1969 . Chisolm , J.J. , Jr. : Aminoaciduri a a s a manifestatio no f rena l tubula

r injur y i n lea d intoxicatio

nan d a compariso

nwit h pattern s o f aminoaciduri a see ni n other diseases . J . Pediatr . 60:1-17 , 1962 . Choie , D.D. , Richter , G.W. : Cel l proliferatio nin ra t kidne

y Induce d b y lea d acetat e an

d effect s o f uninephrectom y o n th e pro liferation . Am . J . Path . 66:265-275 , 1972a . Choie , D.D. , Richter , G.W. : Stimulatio no f DN

A synthesi s i

n ra t kidne

y b y repeate d administratio

no f lead . Proc . Soc . Exp . Biol . Med . 142:446-449 , 1972b . 

### Cohen , S. , Sweet , A.Y. , Mautner , W. , Churg , J Ligh tand electro n microscop y o f lea d nephropathy . Child . 100:559-56 0 (abs) , 1960 . , Grlsham , E . Am . J . Dis .

Cramer , K. , Goyer , R.A. , Jagenburg , O.R , Wilson , M.H. : Rena l ultrastructure , rena l function'an dparameter s o f lea dtoxicit y i n worker s wit hdifferen t length s o f lea dexposure . Brit . J . Ind . Med. , i npress .

.m.\.r-* • AmmSi?iir.£lliiSm muSmti'im^fP. M

V GOYE R AN D J

. F

. MOOR E 

### -Idne y i n lea d intoxi
-

- Inclusions . Am

.
anaemia , mitochon
11:49-51 , 1965 . •> n bodie s i n kidne y

- Hopkins Hospita l

Ultrastructura l J . Ultrastructura l

Histochemica l odies . J . Path .

Electro nprob e su e o f poisone d

strlbutio no f lead Med . 26:139 -

itestatio no f compariso nwit h J . Pediatr .

tionI nra t kidne y rtomy o nthe pro DN A synthesi s l n |'-'roc . Soc . Exp .

J. , GrIsham , E . Ir . Am . J . Dis . |i.son , M.H. : Rena l lead toxicit y

i n Brit . J . Ind . CELLULA R EFFECT S O F LEA D 45 9
Dallenbach , F. : Di e aufnahme vo nradioactlvem ble i 21 0 durc h di e tubuluseplthelie nde r niere . Verh . Dtsch . Ges . Pathaol . 49 : 179-185 , 1965 . Dingwall-Fordyce , I. , Lane , R.E. : A follow-u p study

o f lea d workers . Br . J . Ind . Med . 20:313-315 , 1963 . Farkas , W.R. : Depolymerizatio no f ribonuclei c aci d b y plumbou s ion . Biochim . Biophys . Act a 155:401-409 , 1968 . Finner , L.L. , Calvery , H.O. : Pathologi c change s i nrat s an d i ndog s fed diet s containin g lead an d arseni c compounds . Arch . Pathol . 27:433-446 , 1939 . Gibson , S.L.M. , Goldberg , A. : Defect s i nhaem synthesi s l n mammalia ntissue s l nexperimenta l lea d poisonin g an

d experimenta l porphyria . Clin . Sci . 38:63-72 , 1970 . Goyer , R.A. : Th e rena l tubul e i nlea d poisoning . I . Mito chondria l swellin g an d aminoaciduria . Lab , Invest . 19:71-77 , 1968 . Goyer , R.A. : Lead toxicity : a proble m i nenvironmenta l pathology . Am . J . Pathol . 64:167-182 , 1971 . Goyer , R.A. : Cellula r an d subcellula r effect s o f lead . Workin g pape r fo r tas k grou p o nmeta l accumulation . Proceeding s o f XVIIt h Internationa l Congres s o f Occupationa l Health , Bueno s Aires , Argen tina , September , 1972 . Goyer , R.A. , Krall , A.R. : Furthe r observation s o nthe mor phology an d biochemistr y

o f mitochondri a fro m kidney s o f norma l an d lead-intoxicate d rats . Fed . Proc . 28:619A , 1969a . Goyer , R.A. , Krall , R. : Ultrastructura l transformatio ni n mitochondri a Isolate d fro m kidney s o f norma l an d lea d intoxicate d rats . J . Cel l Biol . 41:393-400 , 1969b . Goyer , R.A. , Leonard , D.L. , Moore , J.F. , Rhyne , B. , Krigman , M.R. : Lea d dosag e an d th e rol e o f the Intranuclea r inclusio nbody . Arch . Environ . Healt h20:705-711 , 1970a . Goyer , R.A. , Mahaffey , K.R. : Susceptibilit y t o lea d toxicity . Environ . Healt hPerspect . Exptl . Issu e No . 2:73-80 , 1972 . Goyer , R.A. , May , P. , Cates , M. , Krigman , M.R. : Lead and protei nconten t o f isolate d intranuclea r inclusio nbodie s fro m kidney s o f lead-poisone d rats . Lab . Invest . 22:245-251 , 1970b . Goyer , R.A. , Moore , J.F. , Barrow , E.M. : Lead bindin g protei n

tfsg*' aTriWittM^^Pfr***-

-ggrrg^ars^ara ;

46 0

« •A

. GOYER AN D J

. F. M OOR E n. A. CiOYE

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