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# OAR Box 1147 Prepped by Ollie Stewart



## Document

Number: 

# 138 ) IV-E-1 1
Docket Number: 

# A-87-1 1

Printed 4/22/2008 2:42:55PM

Sheet 138 of 242P. 2



### Not e t o Docke t A-87-1 1
O nOctobe r 26 , 1987 , representative s o f th e Moto r Vehicle s Manufacturer s Associatio n(MVMA ) me t wit hscientist s a t th e Environmenta l Protectio nAgenc y t o presen t th e result s o f researc hsponsore db y th e MVMA , mos t o f whic hi s unpublishe dbu t ha s bee nsubmitte dfo r publication . Presentation s wer e mad e b y Joh nSh i Her , B . D . Garg , andMichae l Olso nconcernin g thei r research . A lis t o f th e meetin g attendee s an da cop y o f th e paper s distribute d befor e an

ddurin g th e meetin g i s attached . Submitte db

y

Rober t E . McGaughy

' Octobe r 26 , 198 7
Attachmen tP. 3



### MVM A GASOLIN E RESEARC H MEETIN G OCTOBE R26 , 198 7
Participan t Kar l Baetck e Michae l J . Olso n Rober t McGaughy Graham Chatbur n Kath y Steile n B . 0 . Gar g C . C . Gree n R . T . Pau l

J . Sh i 1le r J . Vosta l Arthu r Chi u Willia m Pepelk o Barbar a Mandul a Affiliatio n 

## EP A Genera l Motor s 

## EP A Ford 

## EP A Genera l Motor s Genera l Motor s Moto r Vehicle s Manufacturer s Associatio n For d Genera l Motor s 

## EP A EP A EP AP. 4

87-31. 3

GASOLIN E VAPO REXPOSUR E PARAMETER S
DURIN GREFUELIN

GOPERATION S

JOHN W . SHILLE R

FOR D MOTO RCOMPAN

Y DEARBORN , MICHIGA

N

FORPRESENTATION

A T TH E 80T H ANNUAL MEETIN GAN D EXHIBITION

O F APCA

Ne w York , Ne w

Yor k
Jun e 21-26 , 198 7

— — ' ' — —*—* "i^T-i

— -rmX*-P. 5



### GASOLIN E VAPO REXPOSUR

E PARAMETERS DURIN

GREFUELIN

GOPERATIONS , J . W . Shiller . For dMotor Company , Dearborn , M I 48121 . Inorder t

o accuratel y

characteriz e th

e potentia l healtheffect s o f exposure t o gasolin

e vapor s durin

g refuelin

g operations , a determinatio no f th

e concentration , tim e duratio

n an dchemica l characteristic s o f exposure pattern s mus t be made . Becaus e availabl e field dat a are limited , theoretica l modelin g o f fue l propertie s becam e necessar

y t o generat e a mor e complet e pictur e o f exposure . Availabl e exposur e dat a indicat e that th e mos t significant parameter s affectin g gasolin

e vapor concentratio nare distanc e fro

m th e fue l tan k opening , win

ddirection , an

dorientatio

no f th

e perso

nfillin

g th e tank . Smal l change s i nthes e parameter s produc e a larg e scatte r i nexperimenta l fiel ddata . Theoretica l consideration s indicat e that exposure t o gasolin

e vapor s shoul d decreas e in th e winte r compare

dt o summe r level s eve

nwithth

e greater volatility o f winter fuels . The compositio no f refueling vapor s consist s mainl y o f th e lighte r component s o f gasoline , C S an

dlower . Exposur e durationi s typicall y 1/ 2 to 1-1/ 2 minute s per refuelin

g event addin

g u pt o a tota l o f abou

t 7 0 minute s pe r yea r pe r vehicl e o naverage . I t i s foun

dtha t th

e chemica l compositio no f refuelin

g vapor i s s o different fro m th e actua l fue l compositio nthat thi s mus t b e considere

d inan

y evaluatio

no f potentia l healtheffects .P. 6

- 2 - 87-31. 3

I . INTRODUCTION Gasolin e Refuelin g Vapor s Regulation s tha t are currentl y I nplac e C

o contro l hydrocarbo n emissions , i ngeneral , focu s o nreducin g non-methan e hydrocarbon s tha t participat e i nozone-generatin g reaction s i nch e atmosphere . Th e U.S . Environmenta l Protectio nAgenc y (EPA ) ha s estimate dtha t i f ch e emission s o f gasolin e vapor s during moto r vehicl e refuelin g operation s a c servic e station s ar e conerolled , i c woul dresul e I nabou t a 2 percen t reduccio ni n tota l hydrocarbo nemissions^- . A numbe r o f strategie s hav e bee nidentifie d t o contro l vapo r emission s durin g moto r vehicl e refuelin g a t gasolin e service stations . On e optio ni s a vapo r recover y syste na c servic e station s whic hreturn s th e vapo r t o the undergroundtan k (Stag e I I vapo r control) . Anothe r optio ni s to stor e an ddispos e o f th e vapo r I nth e vehicl e (onboardvapo r control) . The Environmenta l Protectio nAgenc y i s expecte dto propos e rulemaking o ncontrollin g gasolin e refuelin g emission s i n1987 1 . On e importan t questio ni ntha t rulemakin g proces s i s t o determin e whethe r the direc t emission s o f gasolin e vapo r o r specifi c constituent s o f gasolin e vapo r warran t contro l t o protec t publi c health . I f th e best , availabl e answe r t o tha t questio nindicate s cha t direc t contro l i s no t warranted , the ncontro l o f gasolin e vapo r ma y b e require d onl y i narea s needin g additiona l ozon e control . I f th e bes t availabl e answe r indicate s cha c direc t contro l i s necessary , the na nationwid e contro l progra

m ma y b e Implied . Gasolin e Vapo r Healt hEffect s Moto r vehicl e manufacturer s an dEP A requeste dtha t the Healt hEffect s Institut e (HEI ) review the issue s concernin g the potentia l advers e healt h effect s o f gasolin e refueling vapors . Thes e issue s aros e fro m a two-yea r anima l stud y

whic hfoun dincrease dkidne y tumor s i nmal e rat s an d increase dlive r tumor s i nfemale mice , followin g chroni c exposur e to wholl y vaporize d unleade dgasoline^ . I nresponse , the Institut e sponsore d a nin e mont hstud y o f the existin g dat a an dissue da fina l repor t i n Septembe r 1985^ . Som e o f the fact s brough t ou t i nthe HE I repor t ar e a s follows : The bioassa y study * ha s show

nwholl y

vaporize dgasolin e t o be a nanima l carcinoge nan di nth e absenc e o f evidenc e C

o th e contrary , l c migh t als o b e pruden t t o assume tha t vapor s fro m wholl y

vaporize dgasolin e ar e huma ncarcinogens . Ye t becaus e peopl e rarely , i f ever , encounte r wholl y vaporize dgasoline vapor s durin g th e course o f thei r dail y activities , finding s fro m the bioassa y

stud y d o no t necessaril y

mee t th

e informationa l requirement s for the firs t ste pi nquantitativ e ris k assessment , hazar d identification .P. 7

- 3 - 87-31. 3

A possibl e mechanis m fo r the toxi c respons e observe di n

. mal e rat s wa s identifie d an dsuggest s tha t the male ra t ma y b e uniquel y

susceptibl e t o effect s fro m gasolin e vapor , thu s diminishin g it s relevanc e to humans . Benzene , a constituen C o f gasolin e vapo r an da carcinoge ni nhuman s an drodents , i s no t believe dto hav e contribute dto the increase dincidenc e o f tumor s foun di nth

e roden t bioassa y

studies . Availabl e huma nepidemiologi c evidenc e neithe r negate s no r confirm s th

e interpretatio ntha t gasolin e vapor s ar e a potenCia l huma ncarcinogen . Thus , Ch e actua l magnitud e o f associate dhealt hhazards , i f any , canno t b e estimate daccuratel y withou t additiona l data . Purpos e o f Pape r
A s discusse dabove , th e Healt hEffect s Institut e pointe dou t the nee d fo r additiona l dat a befor e a naccurat e estimat e o f the publi c healt hris k du e t o exposur e to gasolin e vapor s ca nbe , made . Thi s pape r deal s wit hon e Importan t elemen t o f th e healt hassessmen t issue , exposure-dos e parameters . Suc hparameter s includ e chemica l characteristic s a s wel l a s concentratio nlevel s an dtim e duratio no f typica l exposur e patterns . Suc h informatio ni s believe dessentia l to medica l professional s i nmakin g thei r determinatio no f publi c healt hrisk . 2 . EXPOSUR E PARAMETER S Variable s Ther e ar e many

factor s tha t influenc e potentia l exposur e t o gasolin e vapo r constituent s durin g refuelin g operations . Som e o f th e mor e importan t factor s are :

Fue l chemica l component s an drelativ e abundance . Ambien t an

dfue l temperatures . Wind"'spee

d an ddirection . Refuelin g tim

e duratio nan

dfil l rate . Distanc e fro

m pum pnozzl e an

dorientatio no f person . Th e fiel dsamplin g dat a s o fa r availabl e cove r onl y a smal l subse t o f th e rang e o f thes e variables . Therefore , fiel dexposur e dat a wer e combine di nthi s pape r wit hfue l modelin g an dothe r dat a t o hel pgenerat e a mor e complet e pictur e o f potentia l exposure . Fiel dExposure Dat a Dat a take nto describ e exposur e t o gasolin e vapor s durin g actua l moto r vehicl e refuelin g operation s ar e availabl e fro m onl y a fe w studies . I none study , shor t term self-servic e exposur e concentration s wer e measure db

y Clayto nEnvironmenta l Consultant s (CEC ) a t 1 3 gasolin e station s usin g a portabl e sampling apparatu s . Show ni nthe firs t tw o majo r section s o f Tabl e I ar e th e CE C dat a for unleaded , leade dan d- 4 - 87-31. 3

premiu m unleade dfuel , firs t average db y fue l typ e an

dsecondb

y th e geographi c locatio no f ch e servic e station s chose ni ntha t study . Als o show ni nTable I ar e dat a fro m simila r studie s b y employee s o f Shel l Oi l Co. 5 , Baeeella 6 » 7 EPA 8 , Mobi l Oi l Corp. 9 , an dGenera l Motors 1 0 (GM) . Al l o f thes e dae a hav e bee nconverte dC o che sam

e basi s fo r compariso ni n Tabl e I . The following observation s ar e mad e regardin g thes e si xfiel d studies . (1 ) Th e measure daverag e concentration s o f gasolin e vapo r listedi n Tabl e I cove r a larg e rang e fro m a lo w o f 0.0 1 t o a hig ho f 21 8 ppm . Althoug hther e ar e man y factor s tha t influenc e the measure dconcentratio n level , th

e EP A study 8 appear s t o hav e clearl y identifie dsome o f th e mor e significan t variables ; win ddirectio nan ddistanc e fro ©pum pnozzle . Highes t concentration s wer e observe dwhe nch e windfirs t passe dove r ch e gasolin e tank an

dpum pnozzle , blowin g emitte dvapor s directl y towardth

e hydrocarbo ndetecto r o r th e perso nfilling the tan k (calle dparalle l wind assumin g the windi s als o paralle l t o the axle s o f the car) . Lowes t concentration s wer e observe dwhe nthe win dwa s blowin g vapor s directl y away fro m the hydrocarbo ndetecto r o r th e perso nfillin g the tank (calle d revers e paralle l wind) . Th e significan t effec t o f winddirectio nan d distanc e fro

m pum pnozzl e i s illustrate di nFigur e 1 . Bot hChe paralle l an drevers e paralle l win dcondition s ar e illustrated , fo r vapo r concentratio n(logarithmi c scale ) a s a functio no f the horizonta l distanc e fro m the fue l tan

k filling nec k (linea r scale) . ' The 2 foo t distanc e wa s selecce db

y che authors " a s the approximat e locatio no f a nindividua l during a refuelin g operatio n("breathin g zone") . A s the distanc e fro m che fue l tan k fille r nec k increased , th e measure dhydrocarbo nvapo r concentratio nrapidl y decrease da s indicated . Th e dat a i nFigur e 2 have backgroundlevel s subtracte dout . A t th e 2 foo t location , hydrocarbo n concentratio nfor th e revers e paralle l win dwa s mor e tha na facto r o f 200 0 time s les s thanthe paralle l windcase . Th e G M study* " als o foun dwidel y varyin g result s wit hchange s i nthe positio no f th e perso nandwind direction . I nfact , th e dominan t reaso nwh y thei r winte r result s wer e highe r thanthei r summe r values , contrar y to expectations , wa s tha t the perso nfillin g the tank wa s close r t o th e fue l nozzl e i nth e winte r program . (2) ' The'averag e ambien t temperatur e dat a liste di nTabl e I for the CEC , EPA , an

dG M studie s wer e take ndirectl y fro m thei r reports . Th e temperatur e dat a liste dfo r th e othe r studie s ar e 3 0 yea r norma l temperature s fo r th e locatio nan dmont huse ddurin g thei r samplin g prograe^ . Mos t o f the dat a i nTabl e I wer e obtaine dfo r ambien t temperature s abov e 60° F andare no t completel y representativ e o f al l season s an

dgeographi c locations . Furthermore , th e larg e scatte r i nthe dae a du e t o th e dominan t effec t o f difference s i nsamplin g distance , orientation , and.win d direction s make i t difficul t t o discer nCemperaCur e effece s base do navailabl e experimenCa l daea . Figur e 2 i s a nilluscracio n o f che expecCe d CemperaCur e dependence o f refuelin g losses , an dhenc e ambien t vapo r concentration , base do ntheoretica l considerations^>1 3 assumin g simila r tan k an ddelivere dfue l temperatur e fo r variou s fue l volatilit y

level s indicate db y Rei dVapo r Pressur e (RVP , psi) . Th e effec t o f fue l weatherin g als o i s approximate di nthe figur e represente db y vehicl e tank fue l leve l a t the beginnin g o f th e refuelin g event .P, 9

- 5 - 87-31. 3

Base do nche expecte dtemperatur e effec t (Figur e 2) , exposur e to gasolin e vapors , othe r thing s hel dconstant , i s expecte dt o decreas e fro m summe r t o winte r i f ther e i s no chang e i nthe fue l specifications . However , a s "indicate di nTabl e I I (column s 4,5, 7 an d8) , fue l propertie s ar e adjuste db

y supplier s fo r seaso nan dgeographica l location . Thi s adjustment , i npart , help s to Insur e gooddriveabilit y an dengin e starting throughou t the year . Averag e norma l teaperature s o nrecord*- * for eac h stat e als o ar e liste dI nTable I I alon g wit haverage fue l volatilit y data,* ^ RVP . The rati o o f the averag e chang e i nfue l RV P i ngoing fro

® summe r (July ) to winte r (January ) divide db y th e chang e i naverag e norma l temperatur e ove r the sam e periodca nbe use da s a nindicatio no f bot hfue l an dseasona l changes . Thi s rati o for eac hstat e i s liste di nthe far righ t colum

no f Tabl e II . Base donFigur e 2 , however , i f th e ^P/<^> T rati o i s les s tha nabou t 0.1 8 psi/°F , the nwinte r refuelin g emission s an d exposur e woul dbe les s thani nthe summer . The 0.1 8 psi/° F valu e wa s calculate db

y drawin g a horizonta l lin e (n o chang e i nemissions ) betwee n the 8. 9 an

d12. 2 ps i fue l curve s anddividin g thi s pressur e differenc e (3. 3 psi ) b

y th e temperatur e differenc e along th e horizonta l axis . The rati o wil l var y

slightl y

wit hstartin g position . The ^ P/^> T ratio s i n Tabl e I I fo r eac hstat e ar e les s tha n0.1 8 psi/° F wit hth e singl e exceptio no f Lo s Angeles , Californi a whic hha s a relativel y larg e chang e i nsumme r t

o winte r fue l volatilit y compare dt o a rathe r smal l chang e i n temperatur e (Lo s Angele s alread y ha s stag e II) . Therefore , almos t withou t exception , winte r refuelin g emission s andexposure concentration s ar e expecte dto b e smalle r thandurin g the summer . (3 ) Reporte daverage s o f al l availabl e yearl y averag e surfac e win d speeddat a throug h198 4 for 4 3 U.S . cities* * wa s foun dt o hav e a mea n valu e o f 9. 2 mphplu s or minu s on e standar ddeviatio no f l. S mph . T o a firs t approximation , on e woul dexpec t gasolin e vapo r concentratio nt o be proportiona l to the tan k fil l rat e an

dinversel y

proportiona l to wind speed . Becaus e mechanica l turbulenc e i nwindvelocit y i s likel y

t o b e larg e aroundphysica l entitie s i nthe fue l servic e area , no attemp t t o correc t for win

dspee do r win

ddirectio nwa s made . (4 ) Averag e reporte dbenzen e concentratio ndat a als o ar e liste di n Tabl e I alon g wit hthe compute drati o o f gasolin e vapo r t o benzene . The rati o o f gasolin e vapo r t o benzen e depend s upo na numbe r o f factor s includin g fue l benzen e level , gasolin e volatility , an dtemperature . Th e rati o tend s t o be highe r a t lowe r temperature s an dca nchang e b

y a s muc h a s a facto r o f 2 o r mor e ove r the ambien t temperatur e rang e o f th e dat a i n Table I fo r typica l gasolines . Th e GM data* 0 ar e a nexampl e o f this . The . rati o i s lowes t a t hig htemperature , lo w volatility , an

dhig hfue l benzen e content . A s a nexample , a mathematica l fue l model* ^ predict s a gasolin e vapo r t o benzen e rati o o f 9 0 fo r 100°F , indolen e gasolin e ( 9 ps i RVP) , and 2 volume percen t benzene . Becaus e th e dat a i nTabl e I represen t lowe r temperatures , generall y highe r volatilit y fuels , an

di nmany

case s lowe r volume percen t fue l benzen e tha nth e exampl e give nabove , th e ratio s woul d mos t likel y

excee d90 . Excep t fo r the unleade dsample s take ni nPhiladelphi a an dpremiu

m unleade dsample s i nHouston , the gasolin e vapo r to benzen e ratio s for the CE C dat a appea r t o be to o low . Therefore , unles s the CE C stud y wa s conducte dfo r gasolin e station s sellin g significantl y highe r tha nexpecte d

### - 6 

### - 87-31. 3

benzen e conten t (no t ver y likely) , the dat a appea r questionable . A n inquir y wit hCEC* ^ reveale dtha t the reporte dtota l H C an dhenc e the gasoline * vapo r data , ma y b e somewha t lo

w du e possibl y

t o som e missin g ligh t hydrocarbo nspecie s bu t eha e eh e benzen e dae a wer e considere d reliabl e fo r the samplin g condition s used . Thi s explanatio nappear s correct o Th e dae a fro m som e o f ehe oehe e studie s i nTabl e I als o may hav e faile dto measur e al l hydrocarbo nvapor s bu t thi s appear s eo b e muc hles s o f a problem

du e to thei r generall y highe r ratios . Sample s i nthe CE C an d Shel l Oi l progra m

wer e originall y

collecte dusin g charcoa l tube s an d processe di nthe laborator y usin g carbo ndisulfid e extraction . Thi s metho di s know

nt o los e very

ligh t hydrocarbo nspecie s an drequire s a relativel y

larg e sampl e for adequat e detectio nlimits . (5 ) Th e EP A

stud y too kthe mos t car e i ndefinin g the "breathin g zone " samplin g location . However , the selecte dplacemen t o f the sampling prob e i nthe othe r studies , althoug hno t wel l defined , stil l must be considere dbecaus e ther e remain s some questio na s t o wha t locatio nbes c represent s averag e exposure . Becaus e o f thes e uncertainties , on e migh t bas e a nexposur e estimat e o nthe simpl e averag e o f al l dat a I nTabl e I (abou t 6 0 ppm ) However , whe nreferrin g to suc ha nexposur e estimat e on e mus t kee pi nmindtha t the missin g hydrocarbo nspecie s ma y hav e cause dth e estimat e to b e too low . O nthe othe r hand , a noverrepresentatio no f revers e paralle l wind , lo w win dspeed , abov e averag e temperatures , and untypicall y

shor t exposur e distance s ma y hav e biase dsuc ha nexposur e estimat e o nthe hig hside . 3 . EXPOSUR E TIM E Th e duratio no f exposur e i s dependen t upo nthe numbe r o f gallon s o f gasolin e t o be transferre dt o the vehicl e an dthe pumpin g rat e assume dfo r eac hself-servic e fillup . Gasolin e pump s typicall y operat e betwee n8 t o 1 2 gallon s pe r minute . Th e averag e yearl y fue l consumptio nfo r al l moto r vehicle s (car s an

dtrucks ) i s almos t 70 0 gallon s pe r year*& . At a 1 0 gallo npe r minut e fil l rate , tota l averag e exposur e duratio nwoul dbe abou t 7 0 minute s pe r year , o f cours e no t al l a t on e time . Assumin g 1 to 2 trip s t o the servic e statio npe r wee kpe r vehicle , a nexposur e duratio n pe r fil l woul drang e fro m abou t 1/ 2 to 1-1/ 2 minute . Th e EP A selecte dthe 1-1/ 2 minut e tim

e perio dfo r thei r samplin g program 8 

### . 4 . COMPOSITIO N

O F REFUELINGVAPO R Bot htheoretica l computations*^»* ' an drefuelin g vapo r measurements*'s* 8 

### »* 9 

### demonstrat e tha t the compositio no f refuelin g vapo r i s strongl y

dependen t onthe compositio no f the liqui dgasoline , but , a s expected , the vapo r i s compose dprimaril y o f the mor e volatil e component s o f gasoline . Th e chemica l component s foundi ngasolin e encompas s a wide-boilin g rang e (-4 7 t o abov e 400°F ) an drepresen t a larg e numbe r o f homologou s hydrocarbon s derive dfro m petroleum . Suc hmixture s ar e nearl y idea l i n the absenc e o f oxygenate s (alcohols) . A nidea l mixture , b y definition , obey s Raoult' s la w

whic hstate s tha t th e partia l pressur e o f eac h componen t i nth e vapo r phas e I s directl y proportiona l t o it s mola r compositio no r to the numbe r densit y o f componen t molecule s i nthe liqui d fuel . Raoult' s la

w ma y b e describe db

y the equation :P.1 1

- 7 - 87-31. 3

P i ° P i . E i wher e p ^ ° th e partia l pressur e o f componen t i ?l ° the vapo r pressur e o f pur e componen t i m ^ <= > th e mola r o r numbe r fractio no f componen t i i nth e liqui dfue l
I f we divid e bot hside s o f th e abov e equatio nb y the tota l fue l vapo r pressur e an

dmultipl y

b y 100 , the nwe hav e the vapo r mol e percen t o r vapo r concentratio npercen t VC ^ whic hi s give nby :

VC A o 10 0 (P i m t / ( 2 P i taj.) ) Thi s equatio nca nb e use dt o estimat e th e percen t concentratio no f an y particula r componen t i ngasolin e vapo r i f it s fue l mola r concentratio n an dvapo r pressur e ar e known . O f cours e non-idea l mixtures , suc ha s a n alcoho l an

dgasolin e blend , ar e mor e complex , bu t Raoult' s la w ca nstil l provide usefu l preliminar y concentratio nestimate s i nsuc hcase s fo r initia l healt hevaluations , unti l mor e accurat e dat a become available . Figur e 3 illustrate s th e differenc e i ncompositio nbetwee nrefuelin g vapo r an

d liqui dfue l base do ncalculation s usin g Raoult' s law fo r a n unleade dgasolin e fue l (#8532 , 80°F , Referenc e 12) . Fo r convenience , the fue l component s along th e X-axi s ar e ordere drigh t t o lef t i nterm s o f increasin g componen t volatility . Th e figur e illustrate s th e expecte d highe r concentratio no f th e mor e volatil e component s i nthe vapo r compare d to th e liqui dphase . I nthi s particula r case , mor e tha n94 % o f th e ga s vapo r molecule s ar e C 5 an dlighte r hydrocarbon s wherea s thes e sam e component s wer e onl y 3 4 volume percen t o f the liqui dgasoline . At lowe r temperatures , th

e percentag e (bu t no t the absolut e quantity ) o f ligh t hydrocarbon s i ngasolin e vapo r increases . Experimenta l data*°>* ' ar e consisten t wit hth e result s show

ni nFigur e 3 . Therefore , th e compositio n o f gasolin e vapo r differ s significantl y fro m the wholl y

vaporize dgasolin e use d i nanima l studies. *
5 . NATIONAL AVERAG E VAPO RCONCENTRATION EXPOSUR E ESTIMAT E Severa l year s ago , EP A made a nestimatio no f the publi c healt hris k fro m exposur e t o gasolin e vapor s durin g vehicl e refueling.* 0 I ntha t estimate , EP A

too k the arithmeti c averag e o f the reporte dvalue s i nthe CE C stud

y an dderive dexposur e concentratio nlevel s ver y clos e t o thos e liste da s "CE C Overal l Averages " i nTable I (smal l difference s wer e du e to roundin g an

dconversio nfacto r assumptions) . However , the CE C stud y di d no t attemp t t o selec t a rando m sampl e o f servic e station s representativ e o f th e geographic , population , o r seasona l temperatur e distributions , biasin g the exposur e estimat e high . O nthe othe r hand , some missin g ligh t hydrocarbon s ma y

hav e biase dthe estimat e lo w a s discusse dpreviously . Unti l bette r dat a become available , th e 6 0 pp m hydrocarbo nestimate , discusse di nSectio n2 (5) , ca nb e use dwit hcautio nandwit hthe understandin g tha t variation s fro m th e average ca nb e larg e for eac h individua l refuelin g event .

t "-^y g .'" a l •r-.^AA irArr^n^jm.-r^mrmm .r..^- .- 8 - 87-31. 3

6 . CONCLUSION S Base do nthe stud

y containe di nthi s paper , the followin g conclusion s ar e drawn :

Th e orientatio no f the perso nt o winddirectio nan dhi s distanc e fro

m th e pum pnozzl e ar e th

e mos t significan t parameter s affectin g potentia l exposur e t o gasolin e vapors . Othe r thing s being equal , suc ha s winddirectio nan d distance , winte r refuelin g exposure , o naverage , i s expecte dto b

e smalle r thansumme r exposur e wit hfe w exception s despit e highe r volatilit y winte r fuel . Fo r mos t situations , th e rati o o f gasolin e vapo r t o benzen e concentratio nwil l b e greate r tha n90 . Thi s indicate s tha t benzen e concentratio nusuall y wil l b

e abou t I Z (rati o o f 100 ) o r les s o f th e hydrocarbo n vapors . Exposur e t o benzen e during refueling operation s i nth e winte r i s expecte dto b e lowe r no t onl y

becaus e the concentratio no f tota l hydrocarbon s i s lowe r bu t als o becaus e th

e percentag e o f benzen e ca nbe hal f tha t during summe r refueling operation s du e t o temperatur e effects . Refuelin g vapo r contain s mainl y ligh t hydrocarbons , i nmany case s 90+ 2 C 5 an dlighter . At lowe r temperatures , the percentag e o f ligh t hydrocarbon s increases . Ther e stil l remain s som e questio na s t o wha t locatio n bes t represent s averag e exposure . Unti l bette r dat a becom e available , a naverag e hydrocarbo nconcentratio n o f abou t 6 0 pp

m ca nbe use dwit hcaution . Variation s from th e averag e ca nb e larg e fo r eac hindividua l refueling event . Exposur e duratio npe r fil l range s fro m abou t 1/ 2 t o 1 an d1/ 2 minutes . Thi s woul damoun t t o abou t 7 0 minute s exposur e pe r year . Raoult' s la w

i s a usefu l too l t o estimat e th e concentratio no f individua l component s i ngasolin e vapor . Becaus e the compositio no f refuelin g vapo r i s vastl y differen t fro

m wholl y

vaporize dgasoline , on e shoul dno t extrapolat e potentia l healt heffect s fro m on e situatio n t o th e othe r withou t takin g thi s differenc e int o consideration . S o far , thi s recommendatio nha s no t bee n adequatel y

addressed .

JWS.2 0P.1 3



### - 9 - 87-31. 3

REFERENCE S

1 . Statemen

t of Administrato

r Le e Thoma s of th

e Environmenta l Protectio n Agenc y Befor e Th e Subcommitte e o nHealt han

d th e Environment , Committee o

nEnerg y an dCommerce , U.S . Hous e of Representaclves , o n Nationwid

e Ozon e Nonattainment , Washington , D.C , Februar y 19 , 1987 . 2 . A Chroni c Inhalatio nStud y wit hUnleade d Gasolin e Vapor , H . N . MacFarland , et al. , Journa l o f th e America nColleg e of Toxicology , Vol . 3 , Numbe r 4 , 1984 . 3 . Gasolin

e Vapor Exposure an d Huma nCancer : Evaluatio no f Existin

g Scientifi c Informationan d Recommendation s fo r Futur e Research , Repor t o

f th e Institute' s HealthRevie w Committee , HealthEffect s Institute , September , 1985 . 4 . Clayto

nEnvironmenta l Consultants , Repor t o na Gasoline Exposur e Stud y fo r th e America

nPetroleu

m Institute , CE

C Jo bNo . 18629-15 , draf t copy , Augus t 25 , 1983 . 5 . Servic e Statio nAttendants ' Exposure t o Benzen

e an d Gasolin

e Vapors , McDermot t an

dVos . J. , Shel l Oi l Company , Am . Ind . Hyg . Assoc . J . 40 , 315-321 , April , 1979 . 6 . Battelle-Columbu s Laboratorie s (1977) , a s cited inp . 115 , of Referenc e 7 . 7 . Mara an

dLee , Assessment o f Huma nExposure s t

o Atmospheri c Benzene , EPA-450/3-78-031 , 1978 . 8 . Sel f Servic e Statio nVehicl e Refuelin g Exposur e Study , Andrew E . Bond , Vinso nL . Thompson , an d Gordo nC . Ortman , EP A Environmenta l Monitorin g System

s Laboratory , an

d Franci s M . Black an d Joh nE . Sigsby , Jr. , EP A Atmospheri c Science s Researc hLaboratory , Jun e 1985 . 9 . Gasolin e Vapo r Exposure s a t a Hig hVolum e Servic e Station , C A . Kearne

y an d0 . B . Dunham , Mobi l Oi l Corporation , Am . Ind . Hyg . Assoc . J . 4 7 (8) , 535-539 , 1986 . 10 . Measurement of Vapor Exposure Durin g Gasolin

e Refueling , SA

E pape r #860087 , G . Tironi , G . Nebe l an d R . Williams , presented a t th e Society o f Automotive Engineer s 198 6 Internationa l Congres s an d Exposition , Detroit , Feb . 24-28 , 1986 . 11 . The Worl dAlmana c an dBoo k of Facts , 1986 , Newspape r Enterpris e Association , Inc. , Publishe d fo r th e Detroi t News , 1986 .P.1 4



### - 1 0 - 87-31. 3

12
. Mathematica l Prediction s o f th e Effect s o f Gasolin e Compositio n o n Rei d Vapo r Pressure , Refuelin g Emission s and Thei r Reactivity , J . W . Shiller , SAE pape r #860532 , presente d a t th e Societ yo f Automotiv

e Engineers ' 198 6 Internationa l Congres s an d Exposition , Detroit , Feb . 24-28 , 1986
. 13
. Technica l Report , Refueling Emission s fro mUncontrolle d Vehicles , D . Rothma n an

d R . Johnson , EPA-AA-SDSB-85-6 , Augus t 1985
. 14
. MVM

A198 5 winte r an

d summe r Gasolin e Surveys . 15
. Phon e communication , E . C . Su , Ford , an d Mr . Walcott , Clayto n Environmenta l Consultants , February , 1985
. 16
. MVM

AMoto r Vehicl e Fact s an d Figure s '86
. 17
. Compositio n o f Vapo r Emitte d Fro ma Vehicl e Gasolin e Tan k during Refueling , R . L . Fure yan d B . E . Nagel , SA

E pape r #860086 , presented a t th e Societyo f Automotiv e Engineers ' 198 6 Internationa l Congres s an d Exposition , Detroit , Februar y24-18 , 1986
. 18
. Ques t fo r a Gasolin e TLV , H . J . McDermot t an d S . E . Killiany , Jr. , Am . Ind
. Hyg . Assoc . J . (39) , February , 1978
. 19
. Gasolin e Vapo r Exposur e Durin g Bul k Handlin g Operations , C . F . Phillilp s an d R . K . Jones , Am . Ind
. Hyg . Assoc . J . (39) , February , 1978
. 20
. Estimatio n o f th e Publi c Healt h Ris k fro mExposur e t o Gasolin e Vapo r Vi a th e Gasolin e Marketing System , EPADraf t Staf f Pape r Submitte d fo r revie wt o the Scienc e AdvisoryBoard b yOHE Aan

d OAQPS , EPA , June , 1984
. 21
. Moto r Vehicl e Manufacturer s Associatio n U.S . Gasolin e Survey , Summe r 1983
.

JWS.20 CP.1 5 

### 87-31. 3 Tabl e I - Refuelin

g Studie s of Short Ter m (Fe w Minutes ) Exposure t o "Breathin

g Zone " Gasolin

e Vapo r

Study/Date/Fuel/Locatio n *CEC Overal l Average s (Aug.,'83)(1 ) Unleade d Leade d Premiu m Unleade

d

Philadelphi a (8l°F , 10. 5 mph ) Mariecca , Ga . (9l°F , 1 0 mph ) AClanc a (81°F , 5. 7 mph ) Monterey , Ca . (75°F , 5 mph ) Carmel , Ca . (66°F , 8 mph ) Salinas , Ca . (70°F , 7 mph ) Bellaire , Tx . (92°F , 7 mph ) Housto n(87°F , 10. 3 mph ) *Shel l Oi l Compan

y (2 ) Housto n(May , '77 ) (75°F ) N . Britain , Ct.(Jul,'77 ) (73°F ) N . Orlean s (Jun , '77 ) (80°F ) *Battell e (3 ) *EP A Raleigh , N.C . (4 ) (Feb. , '85 ) Averag e Gasolin e Vapor/pp

m

1 

### 56.5 * 70.5 * 66.2 * ied/Premiu

m i 89/129/- * 36 / 51/- * 61 / 70/73 * 20 / 19/- * 34/-/51 * 62 / 47/- * 80 / 16/55 * 48 / 82/68 *
54. 4 40. 0 77. 3 Averag

e Benzen e (ppm )

0.9 6 1.4 2 0.8 1 

# ( D 0.63/1.9/ - 0.61/0.97/ - 1.12/1.28/ 0 0.57/0.94/ - 1.2/-/1.4/ - 1.8/1.1/ - 1.6/0.57/0.' , 0.71/1.94/ 0
0.1 1 0.4 6 0.9 6 Rati o o f G . Vapor t o Benzen

e


### 59 5 0 8 2

141/68/ - 59/53/ - .8 4 54/55/8 7 35/20/ - 28/-/3 6 34/43/ - 74 50/28/7 4 .6 1 . „ 68/42/11 1
49 5 • 

### 8 7 8 1 Benzen

e 

### i n Gasoline/ %

-

- 

### (7 ) 1.1/.9 3 1.8/.6 3 1.5/.9 6 1.3/1. 3 .97/.8 8 .43/.3 8
(2) ' 

### (2 ) (2 ) E . G a N . 

# s . S . N . Pa . m 

### Ca . Ca . Tx . Tx .

0.2 7



### 24 * Paralle l Win

d(57°F , 4. 5 mph ) (13. 1 RVP ) Revers e Paralle l (55° , 6. 5 mph ) 0.01 * (12. 9 RVP

) 0.2 3 10 4 2.0 2 o f Carbo n 1.9 % o f Carbo n
*Mobi l Oi l Corporatio n (5 ) E . Pennsylvani a (May , '83 ) (63°F ) ~ 

### 2 9 •Genera l Motor s Detroi t Area (6 ) (Aug./Sept . '85 , 64'F , 10. 4 RVP ) 57 * (Feb.'85 , 19°F , 13. 7 RVP ) 218 * 0.5 0 0.9 7 11 4

22 4 3.2 Z o

f Carbo n 3.2 Z of Carbo

n (1 ) Compute

dfro m th e dat a i

nReferenc e 4 . (2 ) Take

nfro m Referenc e 5 . 90 Z of gasolin e sample s ha

d benzen

e conten

t < - I Z (Leaded) , < - 1.3 Z (Unleaded) , an d < - 1.5 Z (Unleade d Premium) . (3 ) Takenfro

m Tabl e IX- 4 o

f Referenc e 7 . (4 ) Takenfro

m Referenc e 8 . Paralle l an d Revers e Paralle l refer t o th e win d direction , either towar

d(parallel ) o

r awa y fro m (revers e parallel ) th e perso nfillin

g th e tan k withreferenc e t

o th e vapo r source . (5 ) Take

nfro m Referenc e 9 , correcte d t o 2 minut e exposure time . (6 ) Take

nfro m Referenc e 10 . (7 ) Average benzen e conten

t i n1983-summe

r gasoline , MVM

A gasolin

e survey , vol Z , Ref . 21 .
* Th e conversio

nt o pp m wa s mad

e assumin

g 4. 6 carbons pe

r molecule , a

naverag

e molecular weigh t o f 6 6 an da hydrogen/carbo

nrati o o

f 2. 4 fo

r gasolin

e vapor .

# FIGUR E 1

Hydrocarbo nVapo r Concentratio nMeasure dDurin g Vehicl e Unleade dGasolin e Refuelin g Operation s

•=^ & 

## Paralle l Wind 4. 5 mp hVelocit y 

# O . Revers e Paralle l Wind . 6. 5 mp

h 

# —O — Win d blowin g vapor

s towar d detecto r Win d blowin g vapors awa y fro m detecto r
012345678. 9 Horizonta l distanc e fro m fue l tan k opening , fee t Note : Th e 2 foo t horizonta l distanc e (5 fee t abov e ground ) wa

s selecte d a s th e approximat e locatio n o f th e breathin g zon e durin g refueling

. 1 0

T J

# FIGUR E 2

Calculate dRefuelin g Los s Versu s Temperatur e Assumin g Tank An

dAdde dFue l A

t Sam e Temperatur e Refueling Loss . Gran s Per'Gallo n

9 r 8 7 6 5 4 3 2 1 RV P 12. 2 —

40 XFul l ,QRV P 12. 2 —

10 % Ful l •~G

-



# ^r* —2&r« •*"-"

' o^iV ' RV

P 10. 5 —

10 % Ful l

RV P 8. 9 — 40 % Ful l

m^mWm-mm^mmm^m * ^MM » 

# RV P 8. 9 — 10 % Ful l



# — £ r —

... . i... . i... . i. . i . i... . i... . i... . i... . i....i... . i....i....i....i....i....i.... i 

# $*$> *$> 4> & &<$>4>&4><p4>#4><fm.4> tt 

# $ Fue l Temperature . Degree s F Referenc e 12 . 

# P

# FIGUR E 3

Relativ e Abundanc e O f Gasolin e Refuelin g Vapo r Component s Compare dT o Liqui dFue l Compositio n Vapor/Fue l

4 0 3 5 3 0 2 5 2 0 1 5 1 0 

## 5 0 volum e percen t

1



# 1 I x 1 '^K

^ 1 fl K

' • H X m-TrH

B S B ( 

# EL &

<> r<> m<> v O -v O V ^ , C V< > , 0 V ^ rS> V O 

## K V

## «A V

## <A V

## A v t 

## O \ ^ A % 

## A % 

## \\>\0\0\v>\\>\0'\0'\K v 

## ^ v 

## ' - V 

# oV^oO ^ <$ > 

# 0 % V J * v V J % <-." •C P 

## r? > r° > K<

> 

## ° * 

# Increasin g volatilit

y Vapo r Volum e %
Fue l Volum e %

Fue l #8532 . 8 0 degree s F , Referenc e 12 .

T J c oSft C

Alabam a Alask a Arizon a Arkansa s Californi a Colorad o Connecticu t Delawar e Florid a Georgi a Hawai i Idah o Illinoi s Indian a Iow a Kansas Kentuck y Louisian a Main e Marylan d Massachusett s Michiga n Minnesot a Mississipp i Missour i Montan a Nebrask a Nevad a Ne wHampshir

e Ne wJerse y Ne wMexic o Ne wYor k Nort h Carolin a Nort h Dakot a Ohi o Oklahom a Orego n Pennsylvani a Rhode Islan d Sout h Carolin a Sout h Dakot a Representativ e Citie s Mobil e Junea u Phoeni x Littl e Roc k Lo s Angeles , Sa n Francisc o Denve r Hartfor d U i Islingto n Jacksonville , Miam i Atlant a Honolul u Bois e Chicag o Indianapoli s Des Moine s Wichita , Kansas Cit y Louisvill e Ne wOrlean s Portlan d Baltisnr e Bosto n Detroit , Saul t Ste . Mari e Him-St . Pau l Jackso n St . Loui s Helena , Billing s Oaah a Reno , Las Vegas Concor d Atlanti c Cit y Albuquerque , Ne wYork , Alban y Charlotte , Raleig h Bismar k Cincinnati , Clevelan d Oklahom a Cit y Portlan d Philadelphia , Harrisbur g Bloc k Islan d Charlesto n Huron , Rapi d Cit y t
Stat e 198 0 Populatio n Fractio n 1.7 2 0.1 8 1.2 0 1.0 1 10.4 5 1.2 8 1.3 7 0.2 6 4.3 0 2.4 1 . 0.4 3 0.4 2 5.0 4 2.4 2 1.2 9 1.0 4 1.6 2 1.8 6 0.5 0 1.8 6 2.5 3 4.0 9 1.8 0 1.1 1 2.1 7 0.3 5 0.6 9 0.3 5 0.4 1 3.2 5 0.5 8 7.7 5 2.6 0 0.2 9 4.7 7 1.3 4 1.1 6 5.2 4 0.4 2 1.3 8 0.3 0 TABL E 1 1 '/Winte r unleade d Regula r Gasolin e an d Jeappratur e Dat a b y Stat e Suaae r (July ) Gasolin e Clas s AST N 043 9 C/ B 0 s : A B B/ A B/ A C C C C/ B C B C/ B C B/ C B C C/ B C 

### c c c c C/ B B B B B/ A C C A C C/ B B C 8 C/ B C C C/ B B Unleade d •

SUM - C1985 ) <Juty > Fue l Volatilit y (osi . RVP

)
11. 1 12.7(u ) 8.3 * 10. 7 8.9 * 10.2 * 11. 5 11. 1 11.4 * 10.3 * 11. 6 10. 3 12.6 * 12. 6 12. 6 10.7 * 11. 6 11.1 * 11. 5 11. 1 11.5 * 12.0 * 11.3 * 11. 1 10.4 * 10.3 * 10. 7 8.6 * 11. 5 11. 1 9.5 * 11.5 * 11. 4 10. 3 12.1 * 10. 7 11. 4 11.1 * 11. 5 10. 3 10. 3 Suaae r

(July ) Averag e

8 2 5 6 9 2 8 2 6 8 7 3 7 3 7 6 8 2 7 9 8 0 7 5 73 7 5 7 6 8 2 7 8 8 2 6 8 7 7 74 6 8 7 3 8 2 7 9 6 8 7 6 7 0 7 0 7 4 7 9 74 7 8 7 0 7 4 8 2 6 8 7 6 7 0 8 2 7 4 " Winte r

(January ) Gasolin e Clas s ASTND43 9
0 E D E/D E/D E E E D D C E E E E E E 0 E E E E E D E E E E/ D E E E/ D E E/ D E E E/D E E E D E Unleade d •

Winte r (1985 ) (January ) Fue l Volatilit y (osi . RVP

)
12. 7 14. 6 12.5 * 12. 7 12.2 * 13.4 * 14. 0 14. 6 12.8 * 13.0 * 11.6(8 ) 14. 1 14.9 * 14. 9 14. 9 14.7 * 14. 5 12.7 * 14. 2 14. 6 14.2 * 15.3 * 14.8 * 12. 7 14.4 * 14.1 * 14. 7 12.6 * 14. 2 14. 6 13.3 * 14.0 * 12. 7 14. 1 15.2 * 12. 7 14. 6 14.6 * 14. 2 13. 0 14. 1 Winte r

(January ) Averag e Teaperatur e 5 1 2 2 5 2 4 0 5 3 3 0 2 5 3 1 6 0 4 2 7 3 3 0 2 1 2 6 1 9 3 0 3 3 5 2 2 2 3 3 3 0 1 8 1 1 4 6 2 9 1 8 1 9 3 2 2 0 3 4 3 5 2 7 4 0 7 2 7 3 6 3 9 3 1 3 1 4 9 1 6 

### A ? 

## A* i
mSmmmi-tk 0.05 2 0.05 6 0.10 5 0.04 8 0.22 0 0.07 4 0.05 2 0.07 8 0.06 4 0.07 3 0.00 0 0.08 4 0.04 4 0.04 7 0.04 0 0.07 7 0.06 4 0.05 3 0.05 9 0.08 0 0.06 1 0.06 6 0.05 6 0.04 4 0.08 0 0.07 6 0.07 0 0.10 5 0.05 4 0.08 8 0.08 6 0.05 3 0.03 4 0.06 0 0.06 6 0.04 3 0.11 0 0.07 8 0.06 9 0.08 2 0.06 6TABL E 1 1 (Continued )

Stat e Tennessee Texa s Uta h Vermon t Virgini a Was h ingto n Wes t Virgini a Wisconsi n Wyoming Nat . Capito l Representativ e Citie s Nashvill e Amarilo , Galvesto n Houston , Sa n
: Antonio , Dalla s Sal t Lak

e Cit y Burl i ngto n Norfol k Seattte-Tacoma , Spokan e Huntingto n Madison , Milwauke e Cheyenn e Washington , D.C . i
Stat e 198 0 Populatio n Fractio n 2.0 3

, 6.2 8
0.6 4 0.2 3 2.3 6 1.8 2 0.8 6 2.0 8 0.2 1 0.2 5 Stmne r

(Juty ) Gasolin e Clas s AST M D43 9 C/ B B/ A 

### i B/ A C C C/ B C C B C Unleade d •

Sterne r (1985 ) (July ) Fue l Volatilit y (esi . RVP

)
10. 3 10.7 *
10. 2 11. 5 11. 1 11.4 * 11. 1 12. 6 10. 2 11.1 * *

Summe r (July ) Averag e Te»ger*,tyr e
7 9 8 2
7 8 7 0 7 8 6 7 7 6 7 1 6 9 7 6 Winte r

(January ) Gasolin e Clas s ASTRD43 9 E/D 0
E E E E E E E E Unleade d •

Winte r (1985 ) (January ) Fue l Volatilit y fosi . RVP

)
13. 0 13.0 *
13. 4 14. 2 13. 8 14.6 * 13. 8 14. 9 13. 4 13.8 * Winte r

(January ) Ave r mat Temperatur e
3 7 4 7
2 9 1 7 4 0 3 2 3 3 1 7 2 6 3 1 

# 4 :

Smil^l
0.06 4 0.06 6
0.06 5 0.05 1 0.07 1 0.09 1 0.06 3 0.04 3 0.04 6 0.06 0 Tota l 100.0 0

•Referenc e 14 . State s wit h a n asteris k (* ) ar e covere d i n th e fue l survey . Value s give n fo r state s no t surveye d represen t th e a nearb y surve y result . Th e averag e temperatur e dat a ar e norma l temperature s o n recor d fo r th e thirty-yea r perio d 195 1 t o 198 0 inclusiv e fo r th e months , an d fo r th e representativ e citie s indicated , referenc e 11 .

JUS.20 0P.2 1



# Th e Engineering Resource

For Advancing

Mobility

40 0 COMMONWEALT H ORIV E WARRENOALE

. P A1509 6 

# SA E Technica l Pape rSerie s

86053 2 

# Mathematica l Predictio n o fEffect s o

f Gasolin e Compositio n o n Rei d Vapo r Pressure . Refuelin g Emission s an d thei r Reactivity J . W . Shille r Automotiv

e Emission s an d Fue l Econom y Office For d Moto r Co .

Internationa l Congres

s an d Expositio n Detroit , Michiga n Februar y 24-28 , 198 6P.2 2


# 86053 2 

# Mathematica l Predictio n o fEffects o

f Gasolin e Compositio n o n Rei d Vapo r Pressure , Refuelin g Emission s an d thei r Reactivit y J . W . Shille r Automotiv

e Emission s an d Fue l Econom y Offic e Ford Moto r Co .

ABSTRAC T Concer n ove rth e Impact s o f change s l n gasolin e compositio n o n refueling , emission s stimulate d asearc h fo rasimpl e techniqu e t o provid e estimate s o f relate d fue l parameters . Accordingly , amathematica l compute rmode l wa s develope d base d o n th e assumptio n tha tgasolin e liquid-vapo requilibri aca n b e predicte d wit h reasonabl e accurac y fro m Raoult' s la wfo rIdea l mixtures . Gasoline - fue l parameter s suc h a s vapo rpressur e (Includin g Rei d Vapo rPressure , RVP) , refuelin g loss , O H reactivit y i n formin g atmospheri cozon e pollution , an d othe rfue l characteristic s wer e addressed . Th e assumption s mad e an d mode l result s wer e validate d b y compariso n t o measure d fue l parameter s fro m 9 unleade d gasolin e fuels . Extrapolation s wer e mad e t o Illustrat e fue l temperatur e an d vapor/liqui d rati o effects . Calculate d RV

Plevel s wer e foun d t o b e withi n 5 % o f measure d value s fo rth e fuel s evaluated . A complete - listin g o f th e model' s sourc e cod e written,speciflcall y fo raFortun e 32:1 6 microcompute rI s provided .

OVERVIE W - Befor e emissio n control s o n automobile s wer e established , th e amoun to f gasolin e vapo rlos tdurin g refuelin g operation s wa s onl y asmal l fractio n (les s tha n 3Z ) o f th e tota l vehicl e hydrocarbon s emitted . (*• ' However , a s th e contro l requirement s o n exhaus t an d evaporativ e emission s wer e progressivel y mad e mor e stringent , refuelin g emission s a s a calculate d percentag e o f tota l vehicl e emission s becam e large ri n compariso n althoug h no tI n magnitude . Du e t o th e difficult y experience d b y anumbe ro f location s l n th e Unite d State s i n th e l Q 70' s an d earl y 1980' s I n tryin g t o achiev e th e ambien tozon e ai rqualit y standar d throug h hydrocarbo n control s b y mandate d Clea n Ai rAc tdeadlines , th e potentia l fo ran y additiona l hydrocarbo n reductio n becam e o f majo rconcer n fo rsom e states . Becaus e o f this , al l hydrocarbo n sources , includin g refuelin g emissions , hav e com e unde rscrutiny . A n importan tfacto rt o conside ri n evaluatin g refuelin g emission s I s th e compositio n o f

commercia l gasoline . Gasolin e compositio n ha s bee n changin g ove rtim e i n respons e t o anumbe ro f factors . Fo rexample , th e additio n o f

butan e ( arelativel y volatil e hydrocarbon ) t o gasolin e ha s becom e economicall y attractiv e a s butane' s marke t pric e ha s bee n lo wl n relationshi p t o th e pric e o f gasoline . Additio n o f butan e Increase s octane , decrease s anerg y conten tan d Increase s fue l volatility . Figur e I I s ar tillustratio n o f th e historica l Increas e I n th e averag e volatilit y o f

summerunleade d regula rgasolin e FIGUR E 1 SUM P 

### Gasolin e Volatilit y Trend . MVM

A Surve y Averag e RVP O f Unleade dRegula r Gasoline s Rei d ViparPressure . 1 1 r

1 0 ps i

9 -

•Number s i n parenthese s designat e reference s a t en d ot paper . 197 8 197 1 19 M 1 9 C i lende rVee r 198 4

0148-719 1 /86/0224-0532S02.5 0 Copyrigh t 198 6 Societ

y ofAutomotiv

e Engineers , Inc .86053 2 P.2 3

fina l equilibriu m vapo rpressur e depend s o n th e temperatur e an d o n th e vapor/liqui d ratio . Th e abov e simplifyin g assumption s regardin g th e gasolin e vapor-liqui d syste m allo wth e vapo ran d liqui d composition , suc h a s I n a n RV P bomb , t o b e calculate d fro m aknowledg e o f th e fue l compositio n an d th e vapo rpressure s o f th e majo rconstituent s i n th e gasoline . Similarly , fo ragive n fue l leve l i n avehicl e fue l tank , th e vapo rcompositio n I n equilibriu m wit h th e gasolin e i n th e tan kan d henc e th e refuelin g losse s ma y h e estimate d fo raspecifie d temperature . Fo rsuc h computation s a n Iterativ e procedur e i s require d t o adjus t componen tconcentration s unti l th e vapor/liqui d equilibriu m i s found . Th e iterattv e procedur e Incorporate d I n th e mode l ensure s convergence , usuall y withi n 1 0 iteration s fo ravapor/liqui d rati o o f

4 . Th e smalle rth e vapor/liqui d ratio , th e faste rth e convergence . Th e genera l vapo rpressur e an d temoeratur e relationshi p use d l n th e mode l i s base d o n publishe d wor kb y va n Krane n an d va n Ne s Thei requatio n relate s th e vapo rpressur e o f a pur e hydrocarbo n t o It s boilin g poin ta s follows : logi o P t wher e 6 07918- 1 io(n 7 * ( 232+B)«(U20-t )

6.07918-3.19837* (U20

_ B ) * ( 232+c )

P t• vapo rpressur e o f th e hydrocarbo n i n ram H g a ttemperatur e ti n degree s C . B « boilin g poin to f th e hydrocarbo n a t atmospheri cpressure , i n degree s C . Althoug h thi s equatio n I s quit e good , t t represent s ageneralizatio n applie d t o al l hydrocarbons . However , whe n ageneralizatio n i s no tessentia l a s asimplifyin g assumption , othe requation s ca n b e use d t o mor e closel y matc h th e behavio ro f individua l gasolin e components . A numbe ro f functiona l relationship s ar e availabl e fo rthi s purpos e (e.g. , Riedel , Antolne" , Frost-Kalkwart-Thodos , etc.^Vapo rpressur e equatlons)^ 1 -^) . r nthi s stud y specia l equation s wer e generate d fo rsom e o f th e mor e volatil e gasolin e component s base d o n publishe d vapo rpressur e an d temperatur e data(">^ ) • Publishe d dat awer e relate d t o a secon d orde rL aGrang e polynomia l fo rth e logarith m o f

pressur e a s th e for m fo rth e selecte d functiona l relationship . Othe r functiona l relationship s als o ar e possible . A separat e equatio n wa s generate d fo reac h componen to f

interest . Suc h specifi c modification s t o th e generalize d va n Krane n an d va n Ne s approac h ar e desirable , particularl y fo rth e mor e volatil e hydrocarbons . Th e L a Grang e polynomia l techniqu e assure s perfec tfi t t o publishe d dat apoint s wit h interpolatio n betwee n points . Th e orde ro f th e polynomia l plu s on e represent s th e numbe ro f point s o f perfec tfit . Thre e dat apoint s fo reac h constituen twer e use d i n thi s study , selecte d t o includ e th e expecte d rang e o f pressure s Importan tfo rtha tconstituen tI n calculatin g In-us e automotiv e gasolin e tan kvapo rpressure . HYDROCARBO

N REACTIVIT Y - Hydrocarbo n emission s ar e controlle d becaus e o f th e rol e the y pla y I n formin g atmospheri cozon e pollution . Individua l hydrocarbo n compounds , however , d o no tal l contribut e equall y t o th e productio n o f

photochemica l smo g an d diffe r significantl y t n thei rpotentia l o rreactivit y t o for m ozone . Th e flr9 teffor tt o appl y a practica l hydrocarbo n reactivit y scal e occurre d I n 196 6 wit h th e implementatio n o f a regulation , know n a s Rul e 66 , b y th e Lo s Angele s Ai rPollutio n Contro l Distric tt o limi t organi cemission s o n th e basi s o f thei r capacit y t o for m photochemica l smo g Sinc e then , othe rreactivit y scale s hav e bee n proposed(l''~21 ) an< i anon-methan e exhaus t hydrocarbo n vehicl e standar d form s apar to f th e Californi aai rqualit y program , o n th e basl 3 tha tmethan e t s essentiall y inert . Reactivit y scale s ar e develope d wit h th e goa l o f determinin g whic h hydrocarbon s ad d t o th e ambien tozone/oxldan tproble m an d whic h ar e o f littl e o rn o consequence . Candidate s t n th e latte rcategor y migh tInclud e methane , ethane , propane , acetylene , benzene , an d methanol . However , alis to f compound s tha tma y b e o f littl e consequenc e fo rsingl e da y ozon e episode s (singl e day s I n whic h ambien tozon e concentration s excee d th e standard ) migh tb e o f som e consequenc e I n multi-da y episodes . I n multi-da y episodes , stagnan tair , lastin g fo r severa l days , trap s emission s an d allow s mor e tim e fo rreaction s t o occu ran d fo rozon e concentration s t o buil d u p befor e significan t dilutio n ca n tak e place . I n an y event , lowe r reactivit y tend s t o reduc e th e rat e o f ozon e formation , suc h tha thighe rambien tozone ' concentration s woul d b e les s likely . T h e mode l develope d I n thi s stud y calculate s photochemica l reactlvtt y o f gasolin e vapo ro n th e basi s o f hydrocarbo n O

H reactio n rat e data . Th e us e o f O H reactio n rat e dat a fo rhydrocarbo n reactivit y classificatio n ha s bee n propose d previously.(-0.21 ) i ncontras t t o othe rscale s base d o n simulate d laborator y yield s o f

ozone , i tfocuse s directl y o n th e actua l Initia l rate s o f photooxldatlon , amajo r rat e controllin g ste p fo rozon e formation . Suc h rat e dat afo rhydrocarbon s o f Interes tar e liste d i n Tabl e 1.(22,23 ) TABL E I O H REACTIVIT

Y RAT E DAT A

Compoun d - M<*r.h*n * Ethan « Propan e Hut t\n** P'*nt.»«* * 2-<nrthyl- 2 butan e reltinn l McttiAn n I B»nr»n e Tolu^n * o-xvlen f [sopren f O H Reactio n Rflf * (cm*-"i n 1 2 . 7 1 . 5 7. . 4 1.7 l.f l fl "... \ . 0 I . ? fs.m 1 . 2 1. 1 9 . 9 ecule"W" 1 )

X I t * X X X < X X X X X X X ,0-1 5 m-i i io-< 2 m-< 2 IO-' 2 n-' i 11-1 2 10-1 2 ,0-1 2 10-1 2 10" " 10-1 1 io-' < O H <« € t o Reactio n A. Relativ e Methan e I 1 4 2 4 0 JOO n « 4* 5 10,OO O 41* . 12 5 H O
' 2 5 1,50 0 1.1B 0 1 2 . IR O86053 2

show n i n Tabl e 4 . Thus , equation s wer e developed , usin g secon d orde rL aGrang e polynomials , a s discusse d earlier, , t o accoun t fo rvolum e an d densit y change s wit h temperatur e fo reac h majo rfue l constituent . Temperatur e correcte d densit y value s ar e use d durin g th e mode l iteratio n proces s i n estimatin g Fue l vapo rpressure . Whe n measure d relativ e volum e dat awer e no tavailabl e fo racomponent , th e relativ e volum e equatio n fo rachemicall y simila rcomponen twa s used .

TABL E 4 RELATIV E V0LU1E S O F VARIOU S COMPOUND S A S

FUNCTION S O F TEMPERATUR E A T ATMOSPHERI C PRESSURE * distinctl y differen tbas e fuel s fo rwhic h measure d fue l parameter s ar e available . MODE L OUTPU

T FORMA

T - Table s A- l throug h A- 6 i n Appendi x A Illustrat e th e forma tFo rth e displa y o f

th e result s obtaine d fo rth e thre e bas e fuel s usin g th e fue l compositio n dat a obtaine d fro m th e CR C progra m (25 ) § afue ^ temperatur e o f

37.8° C (100"F ) an d avapor/fue l rati o o f

4 (use d i n RV

Pcalculations) . I n Appendi x A ther e ar e tw o table s fo reac h fuel , thos e endin g l n od d number s lis tfue l characteristic s whil e thos e endin g I n eve n number s lis tfue l vapo rcharacteristics . A fue l summary als o I s Include d a tth e botto m o f eve n numbere d tables . Wit h thi s background , mode l outpu tresult s wil l no w b e discussed . DISCUSSIO N O F RESULTS

Relativ e Volum e a t Temperature , OeRrec s C Compoun d 0" C "MT' C 5 0 " C 6 0 " C 95' C Octan e I.000 0 Benspn e I.000 0 Methano l I.000 0

* Referenc e 1 4 1.059 5 1:061 0 t.121 0 1.048 3 I.on /

HEA T O F COMBUSTION - Gasolin e heatin g value , expresse d a s BTU s pe rgallon , I s afue l paramete ro f

genera l Interest . Publishe d mola r heatin g values(^ ) wer e Incorporate d int o th e mode l t o calculat e fue l energ y conten tassumin g th e fina l product s o f combustio n ar e gaseou s carbo n dioxid e an d gaseou s wate ra t25" C an d atmospheri cpressure , startin g wit h liqui d constituent s a t25° C an d a tsaturatio n pressure . MODE L APPLICATIO

N FUE L DAT A - T o validat e an d t o Illustrat e th e applicatio n o f the""mode l develope d l n thi s study- f gasolin e fue l compositio n dat aan d specification s wer e obtaine d fro m a Coordinatin g Researc h Counci l (CRC ) sponsore d stud y conducte d a tth e Nationa l Institut e fo r Petroleu m an d Energ y Researc h (NIPER ) specificall y fo rth e purpos e o f determinin g th e fue l benzen e effec to n automotiv e benzen e emissions.^5 ) T n tna C study , detaile d dat a wer e availabl e fo r9 fuels . Th e stud y use d 2 lo w benzen e bas e fuels , eac h o f whic h wer e divide d int o 4 separat e portion s an d spike d wit h differen tlevel s o f benzene , resultin g I n amatri x o f

8 differen tfuel s I n additio n t o a certificatio n referenc e fue l referre d t o a s Indolen e 8523 . Table s A-l , A-3 , an d A- 5 i n Appendi x A contai n th e fue l compositio n dat a (I n Colum n 2 ) fo rth e Indolen e an d th e tw o specia l lo wbenzen e bas e fuel s (25 % aromatic s an d 40 % aromatics ) use d l n tha t3tudy(25) . Thes e dat aar e particularl y usefu l fo rmode l validatio n becaus e the y relat e t o thre e FUE L RV P- Detail s o f

th e RV

Pmeasuremen t a r e describe d I n AST M Metho d D323 . Th e techniqu e report s fue l vapo rpressur e measure d a t37.8° C (100°F ) i n acontaine rhavin g a4 t o 1 rati o o f

ai rt o liquid . Ai rpressur e i s no t Include d eithe rI n th e mode l calculatio n o ri n th e RV Pmeasurement . Figur e 2 compare s th e calculate d fue l RV Pwit h measure d RVPC25 ) ua t n g th e equivalen tmicr o vapo rpressur e techniqu e fo rth e fuel s o f

th e CR

C study . Measure d microvapo rpressure s hav e bee n correcte d t o th e equivalen tRV

Pusin g th e correlatio n metho d describe d I n AST

M D2551 . Fuel s 853 1 t o 853 4 represen tth e 25% aromati cbas e fue l wit h fou r differen tlevel s o f benzene . Fuel s 854 1 t o 854 4 represen tth e 40 % aromati cbas e fue l wit h simila raddition s o f benzene . A s foun d i n Tabl e 5 th e measure d an d calculate d RV Pvalue s a r e withi n 5% . Bot h th e measure d an d calculate d value s Indicat e adecreas e I n RVP '



### FIGUR E 2 Calculate dAndHtasurs dRai dVapo r Praaaur a

Cilculete d RV

P Measure d RV P

Fue l Vapo r Preseure . pi t Fuel e >S3 > t e M M eer e tlenee * fre e tfi e le e ereeatl c eee e OSf l . Fuel a S94 1 t e SS4 4 eer e olenee e fre a tte Me n ereeatl c eee e (40U

.86053 2 BENZEN E LEVEL

S - Figur e 5 compare s measure d fue l tan kbenzen e vapo rlevel s abov e th e liqui d fue l a tatemperatur e o f 85"F(25 ) wit h calculate d values . Ther e i s goo d agreemen t betwee n measure d an d calculate d value s excep t fo rth e lo w benzen e fuels , possibl y du e t o measuremen tInaccuracie s a tth e ver y lo w benzen e levels . REACTIVIT

Y - A o afram e o f

reference , Tabl e 6 list s 2 1 hydrocarbon s tha thav e bee n show n t o accoun to n averag e fo rabou t9 2 percen to f ambien tgasolin e vapor s aroun d marketin g plant s wher e gasolin e I s loade d Int o tan ktruck s fo r deliver y t o servic e statlons(2 - ) . Th e
TABU ! A MEASURE D OASOLIN E VAPO R COMPONENrS , MOL E 7.* 

### FIGUR E 9 Calculate dandMaaaure dFua l TankBanzan a Vapor a Fo r a Fua l Teaperatur e o f 8 5 Degree s F

Compoun d Propan e formal butan e tsobutan e Isopentan e Normal pentan e Cyclopentan e 2,3-dlmethylbutan e 2-methylpentan e 3-nethylpentan e formal henan e Methy l cyclopentan e 2,4-dlmethylpentan e 2,3-dtmethylpentan e 2,2,4-trlmethylpeat a Iaobuty'en e 1-meehyl-l-buccn e cl»-2-penten e 2-methyl-2-buten e Benzen e Toluen e Xylen e (p,m,o )

Total * Correctio n Point , C

- 42. 1

- 0. 5

- U. 8 27. 9 16. 1 49. 1 58. 0 60. 3 63. 1 68. 7 71. 8 80. 1 89. 8 v s 99. 2

- 6. 9 31. 2 17. 0 18. 6 80. 1 no. 6 142. 0

fo rralsstni Reactivit y

240 " 11 8 30 0 48 0 47 5 71 3 76 0 42 5 42 5 61 3 66 3 76 3 76 3 52 5 588 0 625 0 600 0 1000 0 15 0 72 5 270 0

I H C — * * * * * « * * * * * * * * Mol e I 0. 8 38. 1 5. 2 2 2 . 7. 0 0. 7 0. 7 2. 1 1. 6 1. 5 1. 3 0. 4 0. 7 0. 5 1. 1 1. 6 1. 2 1. 7 0. 7 1. 8 0. 5
92. 1

- > Relativ e

Reactivit y 1. 9 129. 8 15. 6 109. 9 33. 3 5. 0 5. 3 8. 9 6. 8 9. 2 8. 6 3. 1 5. 3 2. 6 64. 7 100. 0 72. 0 170. 0 1. 1 13. 1 13. 5
779. 7 346. 6 Reference ' 26 . Estimate dvalue . Mlllora M Benzene Pe r Gallon n o



# • <«««

+ Fue l Identificatio n Muste r an d Benzen

e Volua e X Fuel e * an i t e * 0B3 4 reereeen t th e le a ereeatl c beo e BSD . Fuel , f 094 1 t o I 0S4 4 reereeen t th e Me n ereeatl c bee e I40 H . reactivity

o f thes e hydrocarbon s i s estimate d to b e about 350 . Tabl e "7 summarize s th e vapor reactivity

calculation s mad e fo r th e 9 fuel s o f th e CR C study . Reactivit y estimate s fo

r th e low aromati c specia l fuel s ar e lowe r tha n comparabl e reactivit y foundnea r marketin g plant s wherea s th e reactivit y o f the hig h aromati c fuel s ar e abou t th e same . Th e Indolene fue l use

dI nth e CR C progra

m ha d a significantl y

highe r reactivity du e largel y

t o th e quantit y

o f C 4 olefi ninth e vapo r phas e (Tabl e A-2) . Figur e 6 display s th e O H reactivity

of completel y

vaporize

d fue l a s a

n 

### FI6UR E 6 Fue l TankVapo r AndCoapletel y Vaporize dFua l O H Reactivit y

Relativ e T o Methan e
SUMMAR YO f FUE L CALCULATION S

VAPO R "» •PR'JSSUR* . FUE L (psi )

851 1
USII

651 1

851 4

854 1

854 2

8543 .
854 4

INDOLF.NF . 852 ) 11.1 0

6.4 1 I0.«> 1 8.4 9 10.7 9 8.1 7 10.6 5 8.2 ? 9.5 6 7.4 4 9.4 ) 7.3 1 9.3 2 7.2 4 9.2 2 7.1 6 8.9 0 6.8 9 Fl* L

rr.Mpr.nATime CF )
to o 8 5 10 0 8 3 10 0 8 3 10 0 9 3
10 0 8 5
10 0 8 5
IO O 8 5 10 0 8 5 10 0 8 5 VAPOR /

I.10.UI 0 RATI O VAPO R

REACTIVIT Y (CH f I )

4 7 0 46 7 46 8 46 5 46 6 46 4 46 5 46 2 79 6 80 2 79 2 79 8 78 8 79 5 78 4 79 1 2)8 0 241 0 REFUELIN G LOS S (C/CAL ) Calculate d Measure d

7. 4 5 . 9 7 . ) 3. 8 7 . 2 5 . 7 7 . 1 5 . 6 6 . 3 5 . 0 6. 3 4 . 9 6. 2 4 . 9 6. 1 4 . 8 6 . 0 S.I 6 . 1

6 . 1

6 . 0

5. 9

5. 5

5. 2

5. 3

5 . 5 Coapletel y Vepertze d Fue l Reactivit y Fue l Ten k

Vipe r Reactivit y

Fue l Identificatio n Nuabe r Fuel e # an i t e * SB M ra y won t th e le e ti c Me e OS

O . Fuel e # 054

J t e * 004

4 th e bla h areeetl c eee

e I40S J .86053 2 P.2 6

1 1 . va n Nes , K. , an d va n Westen , H . A. , "Aspect s o f

th e Constitutio n o f Minera l Oils, " Elsevie rPublishin g Co. , Inc. , 1951 , 104 . 12 . Reid , R . C. , Prausnlty , J . M. , an d Sherwood , T . K. , "Th e Propertie s o f Gase s an d Liquids , Thir d Edition, " McGraw-Hil l Boo kCompany . 1 3 . CR C Handboo ko f

Chemistr y an d Physics , 64t h Edition , CR

C Press , Inc . Boc aRaton , Florida , 1983-1984 . 14 . America n Institut e o f Physic s Handbook , Secon d Edition , McGraw-Hill . 1 5 . Brunelle , M . F. , Dickinson , J . E. , an d Hamming , W

. J. , "Effectivenes s o f Organi c Solvent s l n Photochemica l Smo g Formation, " Solven tProject , Fina l Report , Los Angele s Count y Ai rPollutio n Contro l District , Jul y 1966 . 2 3 . Mill , T. , Winterle , J . S. , Davenport , J . E . , "Validatio n o f Estimatio n Technique s fo rPredictin g Environmenta l Transformatio n o f Chemicals, " Fina l Report , EP A Contrac t No . 68-01-6269 , SRI International , Menl o Park , California . 2 4 . Cox , F . W. , "Physica l Propertie s o f Gasoline/Alcoho l Blends, " Departmen to f Energy , BETC/RI-79/4 , Septembe r1979 . 2 5 . Automotiv e Benzen e Emissions , CTC-APRAC Projec tNo . CAPE-35-83 , Preliminar y Report , presente d t o th e Californi aAi rResource s Boar d an d th e U.S . Environmenta l Protectio n Agency , Octobe r8 , 1985 . 2 6 . McDermott , H . J. , Kllllany , Jr. , S . E. , "Ques tFo raGasolin e TLV, " America n Industria l Hygien e Associatio n (39) , February , 1978 .

16 . Altshuller , A . P. , "A n Evaluatio n o f Technique s fo rth e Determinatio n o f th e Photochemica l Reactivit y o f Organi c Emissions, " JAPCA , 16 , 257 , 1966 . 17 . Trijonis , J . C , Arledge , K. W. , "Utilit y o f Reactivit y Criteri aI n Organi cEmissio n Contro l Strategie s fo rLo s Angeles, " TR W Environmenta l Services , Redond o Beach , California , December , 1975 . 18 . "Proceeding s o f th e Solven tReactivit y Conference, " EPA-650/3-74-010 , November , 1974 . 19 . Jackson , M

. W. , an d Everett , R . L. , "Effec t o f Fue l Compositio n o n Amoun tan d Reactivit y o f

Evaporativ e Emissions, " SA E PT-14 , 802 , 1967-70 . 2 0 . Niki , H. , Daby , E . E. , an d Weinstock , B. , "Mechanism s of~Smo g Reactions, " AC S Advance s i n Chemistr y Serie s 113 , 16 , Photochemica l Smog an d Ozon e Reactions , 1972 . 2 1 . Pitts , J . N. , Jr. , Lloyd , A . E. , Winer , A . M. , Darnall , K . R . , an d Doyle , G . J. , "Developmen tan d Applicatio n o f a Hydrocarbo n Reactivit y Scal e Base d o n Reactio n Wit h th e Hydroxy l Radical , APC A Pape r#76-31.1 , 69t h Annua l Meeting , Portland , Oregon , Jun e 27-Jul y 1 , 1976 . 2 2 . Atkinson , Ri an d Lloyd , A.C , "Evaluatio n o f Kineti can d Mechanisti cDat afo r Modelin g o f

Photochemica l Smog, " Journa l o f Physica l an d Chemica l Referenc e Data , Vol . 13 , No . 2 , 315-444 , 1984 .P.2 7

86053 2 1 1

Tabl i A- 3 Fue l Characteristic s Fo r Fue l » 353 1 (25 1 aromatlcs l Calculate d Fue l Vapo r Pressur e o f ll. l psi . o r S73.9 8 • •H 9 A t fue l temperatur e 100.0 0 degree s F , o r 37.7 8 degree s C

Componen t C3H 6 C3H 8 C4H1 0 C4H 8 C4H1 0 CSH1 2 C5H1 0 CSH1 2 C5H1 0 C6H1 4 C6H1 2 C6H1 4 C6H1 2 C6H 6 C7H1 6 C7M1 4 C7H1 6 C7H1 4 C7H 8 C8HI 8 C3H1 6 C8H1 8 C3H1 6 C8H1 0 C9H2 0 C9H1 8 C9H2 0 C9H1 2 10H2 2 10H2 2 C10H 8 10H1 4 UH2 4 11H2 4 UH1 6 Origina l

Fue l Volum e Percen t 0.03 0 0 31 0 3.97 0 0.08 0 1.94 0 3.01 0 0.06 0 25.34 0 0.06 0 1.67 0 0.13 0 0.03 0 O-.02 0 0.02 0 4.63 0 0.00 0 0.01 0 0.02 0 6.50 0 30.55 0 0.00 0 0.00 0 0.03 0 6.96 0 1.33 0 0.00 0 0.00 0 9.12 0 0.39 0 0.00 0 0.00 0 2.9S 0 0.03 0 0.30 0 0.18 0 Origina l

Fue l Mol e Percen t 0.04 8 0.47 7 4.89 9 0.11 1 2.52 4 3.40 2 0.07 3 28.72 9 0.08 3 1.65 3 0.13 6 0.03 0 0.02 3 0.02 9 4.06 7 o.no o 0.00 9 0.02 0 7 .98 8 25.01 7 0.00 0 0.00 0 0.02 7 7.S3 8 1 .34 2 0.00 0 0.00 0 8.86 5 0.26 2 0.00 0 0.00 0 2.49 0 0.01 9 0.00 0 0.13 9 Fina l

Fue l Hol e Percen t 0.03 7 o.ia i 4.51 1 0.10 3 2.38 7 3.35 7 0.07 2 28.54 7 0.08 3 1.66 4 0.13 7 0 03 0 0.02 3 0.03 0 4.11 3 0.00 0 0.00 9 0.02 1 S . 10 5 25.38 1 0.00 0 0.00 0 0.02 8 7.65 6 1.36 3 0.00 0 0.00 0 9.00 7 0.26 6 0.00 0 0.00 0 2.53 0 0.01 9 0.00 0 0.14 1 Fue l

Vapo r Pressur e

- PS I 0.08 1 0.71 5 3.21 1 0.06 5 1.22 3 0.68 7 0.01 0 4.44 0 0.00 8 0.11 1 0.00 8 0.00 2 0.00 1 0.00 1 0.13 9 0.00 0 0.00 0 0.00 0 0.08 4 0.27 8 0 OO O 0.00 0 0.00 0 0.02 0 O.OO S 0.00 0 0.00 0 0.00 6 0.00 0 0.00 0 0.00 0 0.00 1 0.00 0 0.00 0 0.00 0 Percen t

Fue l Vapo r Pressur e 0.73 4 6.44 7 28.93 3 0.58 9 11.01 8 6.19 1 0.09 3 40.00 4 0.07 4 0.99 9 0.07 4 0 01 4 0.00 9 0.00 9 1.25 3 0.00 0 0.00 1 0.00 3 0.75 2 2.50 8 0.00 0 0.00 0 0.00 2 0.18 0 0.04 5 0.00 0 0.00 0 0.05 3 0.00 3 0.00 0 0.00 0 0.01 1 0.00 0 0.00 0 0.00 0 Percen t

Componen t 8T U
C 3 olefi n C 3 n-p«raffi n - - isoparaffi n - olefi n n-par a f f i n isoparaffi n- olefi n n-paraff1 n - - napthen e isoparaffi n - olefi n n-paraffi n - - napthen e aromati c [ 7 isoparaffi n • [ 7 olefi n C 7 n-paraffi n- - C 7 napthen e C 7 aromati c C 8 isoparaffi n- C 8 olefi n- C 8 n-paraffi n - - C 8 napthen e C 8 aromati c C9'isoparaffi n - C 9 olefi n C 9 n-paraffi n - - C 9 aromati c CI O isoparaffi n CI O n-paraffi n - 1 0 napthen e 1 0 aromati c 1 1 1 1 Isoparaffi n C n-paraffi n C I t aromati c - C 0.0 2 0.2 4 3.1 6 0.0 7 1.6 3 2.7 0 0.0 6 22.8 7 0.0 6 1.5 6 0.1 ] 0.0 3 0.0 2 0.0 2 4.4 4 0.0 0 0.0 1 0.0 2 7.1 2 31.1 2 0.0 0 0.0 0 0.0 ] 7.8 1 1 8 6 0.0 0 0 -.0 0 10.7 7 0.4 0 0.0 0 0.0 0 3 4 0 0.0 3 0.0 0 0.2 1 99.89 0 100.00 0 100.00 0 11.10 0 100.00 0 100.0 0

Tabl e A- 4 Fue l Vapo r Characteristic s Fo r Fue l I 653 1 (25 1 aromatics ) Vapo r Gram s Vapo r Fue l Mole-Vo l Vapo r / XVapo r Componen t Percen t ga l vap . Mas s Reactivit y C 3 olefi n C3H 6 0.73 4 0.0346 3 0.46 6 22. 9 C 3 n-paraffi n C3H 8 6.44 7 0.3186 8 4.29 0 15. 5 C 4 isoparaffi n C4H1 0 28.93 3 1.8S54 2 25.38 2 86. 8 C 4 olefi n C4H 8 0.58 9 0.0370 5 0.49 9 34. 6 C 4 n-paraffi n C4H1 0 11.01 8 0.7179 8 9.66 6 17. 2 C S isoparaffi n CSr l 1 2 6.19 1 0.5008 5 6.74 3 29. 7 C S olefi n CSMI O 0.09 3 0.0073 4 0.09 9 9. 3 C S n-paraffi n .--- - C5H1 2 40.00 4 3.2361 9 43.56 7 190. 0 C 5 napthen e CStU O 0.07 4 0.0058 6 0.07 9 O. S C 6 isooaraffi n C6H1 4 0.99 9 0.3965 2 1.29 9 4 2 C 6 olefi n C6M1 2 0.07 4 0.0070 1 0.09 4 6. 5 C 6 n-paraffi n C6H1 4 0.01 4 0.0013 1 0.01 8 0. 1 C 6 napthen e C6H1 2 0.00 9 0.0008 8 0.01 2 0. 1 C 6 aromati c C6H 6 0.00 9 O.OQ07 5 0.01 0 0. 0 C 7 ftoparaffi n C7H1 6 1.25 3 0.1408 4 1.89 6 9. 6 C 7 olefi n C7H1 4 0.00 0 0.0000 0 0.00 0 0. 0 C 7 n.paraffi n C7N1 6 0.00 1 ' 0.0001 5 0.00 2 0. 0 C 7 naptnen e C7H1 4 0.00 3 0.0003 1 0.00 4 0. 0 C 7 aromati c C7H 8 0.7S 2 0.0777 3 1.04 6 5. 5 C S isoparaffi n C8H1 8 2.50 8 0.3212 0 4.32 4 13. 2 C B olefi n C8H1 6 0.00 0 0.0000 0 0.00 0 0. 0 C a n-paraffi n C3H1 8 0.00 0 0.0000 0 0.00 0 0 0 C 8 napthen e --- - C3H1 6 0.00 2 0.0002 0 0.00 3 0.0 . C 8 aromati c C8H1 0 0.18 0 0.0213 7 0.28 8 3. 3 C 9 Isoparaffi n C9M2 0 0.04 5 0.0064 4 0.08 7 0. 5 C 9 Olefi n C9H1 8 0.00 0 0.OOOO O 0.00 0 0. 0 C 9 n-paraffi n C9H2 0 0.00 0 0.0000 0 0.00 0 0. 0 C 9 aromati c -.-- - C9H1 2 0.0S 3 0.0072 0 0.09 7 0. 7 CI O Isoparaffi n C10H2 2 0.00 3 0.0004 8 0.00 6 0. 0 CI O n-paraffi n C10H2 2 0.00 0 0.0000 0 0.00 0 0. 0 CI O napthen e C10H 8 0.00 0 0.0000 0 0 00 0 0 0 CI O aromati c C10H1 4 0.01 1 0.0016 7 0.02 2 0. 2 • C l l isoparaffi n C11H2 4 0.00 0 0.0000 1 0.00 0 0. 0 C-1 1 n-paraffi n C11M2 4 0.00 0 0.0000 0 0.00 0 0. 0 C l l aromati c CUH1 6 0.00 0 0.0000 4 0.00 0 0. 0 TOTAL S 100.00 0 7.4281 0 100.00 0 470. S Th e averag e molecula r aeigh t o f liqui d fue l I s 93.9 1 Fina l 94.3 5 Th e averag e molecula r aeigh t o f th e vapo r i s 66.2 5 Th e origina l vapor/liqui d rati o I s 4 Th e fina l I s 4.0134 8 Th e origina l fue l mas s pe r m l i s .7196 9 Th e fina l .7212 8 Th e aoov e fue l densit y I s calculate d fo r 2 0 degree s C Th e origina l fue l reactivit y relativ e t o methan e * 1 i s 753. 1 Th e refuelin g los s aoul d o e 7.42 6 gram s pe r U.S . gallo n Thi s refuelin g los s I s 112.1 2 millimole s pe r U.S . gallo n Th e liqui d fue l hydrogen/carbo n rati o is - 1.9 7 Th e vaoo r hydrogen/carbo n rati o i s 2.4 1 Th e fue l ST U pe r gallo n i s U217 786053 2 1 3 APPENDI XB 1 re m ** * Vapo r pressur e program , nZ I s 9 of components . 2 re m ** * Line number s optiona l fo r Fortune 32-1 6 excep t goto . 5 nZ-3 5 di m narae$(nZ) , lb(n*' > ,mf(nZ ) ,raw(nZ ) ,vmf(nZ ) ,ovmg ( nX),si(nZ),den(nZ ) di mbl(nZ),lp(nZ,3),vap(nZ,3),pmass(nZ),ml(nZ), p vk(nZ t di m tk(nZ,3),btc(nZ'»,mfe(nZ),rea(nZ'>,fuelr(nZ ) dim pres(nZ>,rvp(nZ>,percent(nZ),react(nZ>,astra t Z(l9),astmp(19 ) di m ract(l9),vract(l9),vmfe(nZ),gra(nZ),volp(nZ ) ,racora(nZ),hn(nZ),cn(nX ) di mtcden(nZ),mhv(nZ ) re m ** * Rea

dspecifi c componen t dat a ** * 30 0 for 1 - I t

o n Z rea d name$(l),mw(l),den(i),hn(l),cn(i),btc(l),react ( l).mhv(l ) next l prin t "Ente r

Fue l Identificatio n name . " ; Inpu t fuelname$:prin t 70 0 Inpu t

"Ente r fue l temperatur e I n degree s F **;temp f 71 0 temp e - 5*(temp f - 32)/ 9 72 0 terap k

- temp e + 273.1 6 72 2 prlnt:print:prln t

r e m****** * DENSIT Y CALCULATION

S ********** * vrv o = 1.031183 7 vtv o - 1.1182172-0.002213587*tempk+(6.5192982e-6)* t empk© 2 fo r l- l t o 1 2 tcden(l)-den(i)*vrvo/vtv o nex t 1 fo r 1=1 5 t o 1 7 tcden(1)-den(1)*vrvo/vtv o nex t 1 vrv o - 1.025063 9 -» vtvo—
- 0.8581784-0.000107003*tempk+(2.2923977e-6)* t erapk© 2 fo r 1-1 3 t o 1 4 tcden(l)-den(l)*vrvo/vtv o nex t I fo r 1-1 8 t o 1 9 tcden(l)-den(l)*vrvo/vtv o nex t 1 fo r 1-2 3 t o 2 4 tcden(l)-den(l)*vrvo/vtv o nex t 1 tcden(28)-den(28)*vrvo/vtv o fo r 1-3 1 t o 3 2 tcden(l)-den(l)*vrvo/vtv o nex t 1 tcden(35)=den(35)*vrvo/vtv o vrvo-l.023572 5 vtvo-0.8803969-0.000197926*tempk+(2.327485e-6) * tempk© 2 fo r 1-2 0 t o 2 2 tcden(1)-den(1)*vrvo/vtv o nex t I fo r 1-2 5 t o 2 7 tcden(l)-den(l)*vrvo/vtv o nex t i fo r 1-2 9 t o 3 0 tcden(l)-den(i)*vrvo/vtv o nex t 1 fo r 1-3 3 t o 3 4 tcden(l)-den(l)*vrvo/vtv o nex t i vrvo-l.0248712 2 vtvo-0.77441457+0.0004929225*tempk+(I.2l875e-6 ) *tempk« 2 input "Ente r ai r volum e fractio

nabov e liquid . Us e 4 fo

r actua l RVP . ";v

o print print "Ar e your dat a mol e percent o r volum e Z ? " 72 3 Inpu t " Ente r m p o r v p > ";qz$:prin t 72 7 print rprln

t i f qz$-"vp " the

ngot o 76 1 i f qz$-"mp " thengot o 73 0 print:got o 72 3 73 0 fo r I - I to n Z 74 0 print "Ente r mol e percen t ";name$(i); " " 75 0 inpu

t " > ";pe t mf(l)-pet/l0 0 76 0 next I fo r I - I t

o n Z mcom(l ) • mf(l ) gra(l ) - mcora(i)*mw(i ) re m * * I nth

e next lin

e volp(l ) - m l o f componen t I fo

r 1 mol e o f al l component s volp(l ) - gra(l)/den(i ) next 1 tomo l • 0 fo r I - 1 t

o n Z tomo l - tomo l + volp(i) : re m * * Tota l ml o f component s next 1 fo r 1 - 1 to n Z volp(l ) - 10 0 * volp(l)/tomol : re m * * Volum

e percen t calculation next I tomo l - 0 fo r 1 - I t

o n Z tomo l - tomo l + volp(i ) next 1 got o 81 0 76 1 fo r I - I t

o n Z • print "Ente r volum e percen t ";name$(l); " " Input > ";volp(l ) next t fo r 1 - 1 t

o n Z gra(l ) - voip(l)*den(i) : re m * * Densit y

a t 2 0 degree s C mcom(l ) - gra(l)/mw(i ) next i tomo l - 0 fo r I - I t

o n Z tomo l - tomo l + mcora(l ) next 1 fo r i - 1 to n Z mf(l ) - mcoia(i)/tomo lP.2 9

86053 2 1 5 82 6 trv p - trv p + rvp(i):nex ti:fo r1 - I c o nZ.-percent(l ) - I00*r'vp(l)/trv p 82 7 nex tI 82 8 tpre s =

• lnt(le2*tpre s +.5)/le 2 82 9 trv p- lnt(le2*trvp+ .5)/le2:lprlnte r widt h120 : re

m * * Fortun e fil e re m * * prin t command - a t specifie dwidt h print:print:prInt:print:prlnt:print:prin t print " Fue l Characteristic s Fo r Fue l ";fuelname$:prln t print " Calculate dFue l Vapo r Pressur e o f ";trvp; " psi , o r ";tpres; " m m Hg " mat2$- " A t fue l temperatur e 9999.99 degree s F , o r 999.99 degree s C " 85 6 prin t usin g mat2$;tempf,temp e 89 0 prin t tmf e - 0:tmfa e - 0 fo r I - 1 t o n Z mfe(l ) - mt(l ) - sl(l) : re m * * Fina l fua l mllllmole s pe r componen t I tmf e - tmf e + mfe(l) : re m * * Tota l fina l fue l mllllmole s nex t I for I - 1 t o n Z mfe(l ) - mfe(l)/tmfe : re m * * Mol e fractio n- fina l fue l tmfa e - tmfa e + mfe(l ) nex t I sumb l • 0:btuga l - 0 fo r 1 - 1 t o n Z mhv(l ) - 3.9685*mf(l)*mhv(i ) gra(l ) - mf(i)*mw(l ) bl(l ) - gra(l)/den(i ) sumb l - sumb l + bl(l ) nex t 1 fo r I - 1 t o n Z mhv(t ) - 3785.4*mhv(i)/sumb l btuga l - btuga l + mhv(l ) nex t 1 prin t " Origina l Fina l Fue l Percent " prin t " Fue l Fue l Vapo r Component " prin t " Mol e Mol e Pressur e prlnt~- " Componen t Percen t Percen t ps i Contribution " prin t "——»—— — ssAjaasaass s matl$-" / 999.999 999.999 999.999 al $ -" / 9.9999 999.999 9999999.99" mat5$-' 7 9.99999 999.999 9999999.9" a2 S - "I 9.999999 999.999 9999999.9" btugal p» 0 94 0 fo r 1 - I t o n Z btu p= 100*mhv(l)/btuga l btugal p= btugal p+ btu p 95 0 prin t usin g matl$ ; / 9r<.999

r / 9.9999 1999.999 I 9.9999 natne$(l).volp(l),l00*mf(l),l00*mfe(l),rvp(l ) ,percent(l),btu p 96 0 nex tI 97 0 lm f - 0:vp p - 0:vpmm » 0:per p • 0:tvm f - 0:tovmg=0: tpmass- 0 98 0 fo r1= 1 t o n

Z 99 0 lm f » lm f + m f (1 ) :v'pp=vp p + rvp(l):vpmm-vpm m + pres(l ) 100 0 per p • per p + percent(l):nex t1 prin t "aaaa-aaa a a a isiasaaaaaMaaa q « a mm aaisi.aa i aaa i a a aaaaia-aa a aaaaaaa a •asasa a a a aaaaaai a • a a aaaaaaa a aa a a .** a a a ' * hhh $ - " TOTAL S 999.999 99.99999 999.999 9999999.9" ahh $ - " TOTAL S 99* .W 999.999 999.999 999.999 999.999 102 0 prin t usin g ahh$;tomol,100*lmf,100*tmfae,trvp,perp,htug a l p 103 5 tvm f » 0:tovm

g - 0:tpmas s - 0 103 0 fo r 1 - 1 t o n Z 104 0 vmf(l ) - si(l)/ s 105 0 ovmg(l) - sl(l)*mw(l)/100 0 106 0 tvmf - tvmf + vraf(l ) 107 0 tovm

g - tovm

g + ovmg(l ) 108 0 nex t l:v

r - 0:frac t - 0 109 0 for I - 1 t o n Z 110 0 pmass(l ) - 100*ovmg(l)/tovm g 110 5 tpmas s - tpmas s + pmass(I ) fuelr(i ) - mf(l)*react(l ) frac t - frac t + fuelr(i ) frac t - lnt(lel*frac t + .5)/le l rea(l ) - vmf(i)*react(l ) v r - v r + rea(l ) 111 0 nex t I 111 5 fo r l- l t

o I5:prlnt:nex t I print " Fue l Vapo r Characteristic s Fo r Fue l ";fuelname $ Origina l Percen t Fue l Fue l Volum e Vapo r BTU " Percen t Pressur e prin t print " Gram s print " Vapo r / print " ga l vap . print"-—- * aaaaaaa a 111 8 fo r l 112 0 prin t Vapo r Fuel "

Z Vapor " Componen t Mas s Reactivity " -aaaa=ia3aaaasa.a=ia.a.aaaaaaa a asaaaaas a a^maaa-m-aaaa-m' 1 t o n Z usin g Vapo r Mole-Vo l Percen t

aaaaaaat a

mat5$;name$(l),lOO*vmf(l),3785.4*ovmg(l)/vat,p m asc(l),rea(l ) 113 0 nex t I print " —

—— — ———aaaaaaasiaaaa s aaaaaa a 114 0 prin t usin g hhh$;I00*tvraf,3785.4*tovmg/vat , tpmass,v r 115 0 avmw l - 0:avmw

v = 0:avmwl f - 0 116 0 fo r I - 1 t o n Z 117 0 we r - mf(l)*mw(i ) 117 2 ww w - mfe(l)*mw(l ) 118 0 wa r - vmf(l)*mw(l ) 119 0 avmw l - avmw l + we r 120 0 avmw

v • avmw v + wa r 120 5 avmwl f - avmwl f + ww wP.3 0



### GMR-575 3

Rapi dPost-Exposur e Deca y o f a - -Globuli nan d Hyaline Droplet s inth e Kidney s o f Gasoline-Treate d Mal e Rat a

Bhagwa nD . Garg an

d Michae l J . Olso

n Biomedica l Scienc e Department Genera l Motor s Researc hLaboratorie s Warren , Michiga

n4.3090-905 8
Willia a F . Demyaa an d alru nK . Ro y Department o f Biologica l Science s Oaklan d Unirersity Rochester , M I 4806 3

Runnin g Title : Recorer

y froa Gasolin

e Nephropath

y Addres s iaj

Correspondence : Bhagwa nD . Garg Biomedica l Scienc e Department Genera l Motor s Researc hLaboratorie s 3050 0 Moun

d Roa d Warren , M I 4809 0 (313 ) 986-165 2

Submitte

d for Publicatio

nin Journa l o

f Toxicolog

y an d Environmenta l HealthP.3 1



### ABSTRAC T Rapi dPost-Exposur e Deca y o f o^-Globuli nandHyalin e Droplet s i nth e Kidney s
o f Cas o line-Treate dMal e Rats . Garg , B.D. , Olson , M.J. , Demyan , W . an dRoy , A.K . (1987) . J . Toxicol . Environ . Healt h Unleade dgasolin e induce s accumulatio no f hyalin e droplet s inepithelia l cell s o f th e rena l proxima l convolute dtubule s onl y i nmal m rats . Synthesi s o f th e mal e rat-specifi c protein , a- -globulin , a constituen t o f rena l hyalin e dropleta , occur s inth e live r andi s unaltere db y gasoline . Rena l a * -globuli n conten t incresse dt o 210 Xo f contro l vslue s afte r a singl e ora l doa e o f gasolin e (2. 0 mL/kg) ; maxima l level s (320 % o f control ) wer e attaine dfollowin g gasolin e administratio nfo r thre e days . Th e conjoin t effec t o f post-exposur e
recovery

andestradio l (inhibito r o f hepati c a . -globuli nsynthesis ) wa s determine di

nmal e rats . Gasolin e alon e (2. 0 mL/kg , p.o. , 9 days ) increase d
rena l a - -globuli n53 0 Xwit hconcurren t proliferatio no f hyalin e droplets . Withi n3 day s o f terminatin g gasolin e administration , rena l a~-globuli n conten t decrease dt o th e sam e leve l a s tha t i nunexpose drsts . However , th e numbe r o f rena l hyalin e droplet s di dno t retur nt o th e pre-treatmen t leve l unti l day s 6 t o 9 o f recovery . Onpost-exposur e day s 3 , 6 snd9 , estradio l treatmen t ( 1 mg/kg , s.e , 4 days , startin g o nth e 9t hday o f gasolin e treatment ) decrease drens l a . -globuli nconten t t o 75X , S9 Xan d48% , respectively , o f ths t inrst s no t receivin g hormon e treatment . Hepati c tt 2u 

### ~ globuli ninestradiol-treate drat s decrease db y 74X , 97 Xand96 % a t th e sam e intervals . Estradio l treatmen t als o appeare dt o increas e th e rat e a t whic h
norma l rena l morpholog y wa s restored . Thus , accumulatio no f o^-gIobu I i nan d hyalin e droplet s afte r subacut e exposur e t o gasolin e i s rspidl y reversibl e an

d apparentl y

dependen t o ncontinuou s exposur e t o gasolin e sndmaintenanc e o f th e
norma l rat e o f hepati c tt

_-globuli nsynthesis .

• 2

-P.3 2

INTRODUCTION Isopsrsffini c hydrocarbon s an dmixture s o f thes e compound s have beenshow n t o induc e a uniqu e nephropath y i nmal e rat s (Phillip s an dEgan , 1984 ; Gar g e t al. , 1986 ; Kanerv a e t al. , 1986 ; Via ue t al. , 1986) . A ninhalationbioassay protoco l conducte da s psr t o f th e toxicologica l assessmen t o f unleade dgssolin e indicate dths t chroni c exposur e csuse dnephropsth y sn dkidney

tumors . The result s o f thi s study , i nwhic hB6C3F . mic e an dF-34 4 rat s wer e expose dt o wholl y volatilize dgasolin e fo r 2 4 months , showe dtha t gasoline vapo r increase d th e incidenc e o f non-neoplasti c rena l pathology an drena l cortica l carcinom a i n mal e rat s onl y

(Busey

an dCockrell , 1984 ; MacFarlan d e t a I. , 1984) . Becaus e o f th e wide-spres duse o f unleade dgasolin e an da trendtowar dgreate r us e o f self-servic e refuelin g (Environmenta l Healt hLetter , 1985) , significan t attentio nha s bee nfocuse donth e possibilit y tha t recurren t gasolin e exposur e
o f human s ma y

resul t i

nadvers e healt heffects . Althoug hth e mechanis m o f th e sexan dspecies-specifi e toxicit y an d carcinogenicit y

o f gasolin e vapor s remain s obscure , resesrc hha s show ntha t i t doe s no t see

m t o ac t a s a rena l carcinoge nb y producin g somati c mutatio n (Conawa y e t al. , 1983 ; Richardso ne t al. , 1986 ; Loury e t al. , 1986a ; Loury e t

a I. , 1986b) . A

nalternativ e hypothesi s concernin g th e tumorigeni e potentia l o f gasoline i s bsse do nth e observatio ntha t followin g short-term exposur e t o gssolin e o r relste d hydrocsrbo nmixtures , th e epitheliu m o f the rena l proxima l convolute dtubule s (PCT ) o f mal e rat s show s evidenc e o f cel l death , exfoliatio n
an drestorativ e cel l proliferatio n(Buse y an dCockrell , 1984 ; Haide r e t al. , 1984) . Immediatel y precedin g thes e events , PC T epithelia l cells , especiall y thos e o f the S 2 segment , sho w a marke dincreas e i nth e numbe r an

dsiz e o f cytoplasmi c hyalin e droplet s (Haide r e t al. , 1984 ; Gar g e t al. , 1986 ; Olsone t

a I. , 1986 ; Shor t e t a I. , 1986) . Further , i t ha s bee nobserve dtha t th e same populatio no f cell s whic haccumulate s hyalin e droplet s subsequentl y show s a

nP.3 3



### increase drat e o f DN A synthesi s (Shor t e t a I. , 1986) . Th e concep t o f chroni c chemica l Iy-induce dcytotoxicit y an dcel l proliferatio na s a precurso r even t i n carcinogenesi s ha s bee ninvoke dt o explai nth e carcinogeni c effec t o f gasolin e (Loury e t si. , 1986b ) a s wel l s s othe r chemicsI s (Zeis s e t si. , 1986) . Interes t inth e potent-isll y advers e healtheffect s o f gssolin e le du s t o develo ps short-ter m mode l fo r examinin g th e toxicit y o f gasolin e r

nrat s (Gar g
e t al. , 1986) . Ou r initia l researc hshowe dtha t a majo r componen t o f gasoline induce dhyalin e droplet s i s th e androgen-dependen t ra t urinar y protei na ~ - globuli n(Olso ne t si. , 1986) . Electro nmicroscopi c studie s hsv e reveste dtha t gasoline-induce dhyalin e droplet s ar e th e ultrastructura l equivalen t o f phagolysosome s (Gar g e t al. , 1986) . Accumulatio no f thi s protei ndu e t o defectiv e rena l lysosoma l degradatio nha s bee npropose ds s s nintegra l even t i n gasoline-induce dcel l deat hi nth e kidne y

(Klos s sndBus , 1985) . W e hsv e attempte dt o understan dif an dho w gasolin e sIter s th e rens l metabolis m o f a . - globuli nandwhethe r suc halteration s contribut e t o th e nephropsthy associate d wit hfue l hydrocarbo nexposure . Thi s repor t describe s th e relationshi pbetwee n durstio no f gasolin e administratio nandth e rena l accumulatio no f a « -globulin . Further , th e rat e o f deca y o f hyalin e droplet s sndaccumulate da « -globuli n afte r terminatio no f gasolin e exposure,wa s determined .



## -4 -P.3 4

MATERIAL S AN

O METHOD S Experimenta l animal s an dchemical s MaL e Fische r 34 4 rat s (CDF R (F-344)/Cr l BR ; Charle s Rive r Laboratories , Wilmington , MA ) weighin g approximatel y 25 0 g wer e usedthroughou t thes e studies . Rat s wer e maintaine di ncondition s consisten t wit hAAALA C recommendation s wit hPurin a 501 2 cho w an dta pwate r freel y availabl e durin g th e entir e period . Fo r time-cours e experiment s rat s wer e administere dphysiologica l salin e (2. 0 mL/kg ) o r a commerciall y availabl e unleade dgasolin e (2. 0 mL/kg ) b y intragastri c intubatio nonc e dail y an dkille d1 8 hour s afte r th e fina l
treatment . I nhyalin e drople t decay studie s rat s wer e treate dwit hgasolin e (2. 0 mL/kg , p.o. , 9 days ) o r gasolin e plu s estradio l ( 1 mg/k g i na propylen e glyco l vehicl e (Ro y e t al. , 1975) , s.e , 4 days , startin g o nth e fina l day o f gasolin e treatment) . Followin g conjoin t gasolin e an dhormon e treatment , animal s
wer e seriall y

sacrifice da t 3 , 6 an d9 day s afte r th e las t dose o f gssoline . Control s receivin g salin e an drat s treate dwit hgasoline slon e wer e als o allowe dt o recove r fo r 3 t o 9 day s afte r terminatio no f treatment . Al l animal s
wer e sacrifice dbetwee n8:0 0 an d10:0 0 am . Radioimmunoassa y

o f 2„ -globuli ni nkidney

an dliver The cytosoli c fraction s o f kidne y an dlive r homogenate s wer e prepare d(Roy , 1977 ) fro

m sample s o f th e perfuse dorgan s (se e below ) an dasssye dfo r <-*- globuli nb

y double-antibody

radioimmunoassay a s describe db y Ro y (1977) . Th e quantit y

o f < U-globuli ni nth e cytosoli c fractio nwa s expresse drelativ e t o tota l protei nwhic hwa s determine db y th e metho do f Lowry e t al . (1951) . Histologica l procedure s Eightee nhour s afte r th e fina l dos e o f gasolin e o r afte r th e perio do f
recovery , rat s were anesthetize dwit ha nintraperitonea l injectiono f pentobarbital , perfuse dvi a th e hear t wit hcol d0.9 % sodiu m chlorid e solutio n
an dth e kidney s an

dliver s wer e removed , fixed b y immersioni

n10 % neutra l

-5 -P.3 5



### buffere d formalin , processe d routinel yan d embedde d i n paraffin
. 4 /u n section s
o f paraffin- embedde dkidne ywer e staine d wit h Mallory' s Heidenhai n metho d fo r visualizatio n o f hyalin e droplets
.



## -6

-P.3 6



### RESULT S Dependenc e o f rena l flp-globuli naccumulatio nonduratio no f gasolin e adminJstratio n Experiment a wer e conducte dt o determin e whethe r rena l accumulatio no f a - 2 u globuli nresulte dimmediatel y upo ninitiatio no f gasolin e administratio no r require dmultipl e treatments . Mal e rat s wer e treate dorall y wit hgasolin e (2. 0 mL/kg ) onc e dail y

fo r 1 , 3 , 5 , 7 o r 9 days ; 1 8 hour s afte r dosing , live r and kidne y tissue s wer e collecte dfo r determinatio no f a « -globuli nconten t and histologi c evaluation . Followin g a singl e dos e o f gasoline , rena l a - -globuli n level s wer e increase dt o 210 % o f contro l value s afte r 1 8 hour a (Tabl e 1) . Maxima l elevatio no f a . -globuli nconten t (320 % o f control ) inth e kidne y
occurre dafte r 3 day s o f gasolin e administration . Continue dtreatmen t maintained , bu t di dno t significantl y alter , th e elevate dleve l o f rena l a * - globuli n(Tabl e 1) . Th e hepati c conten t o f a . -globuli nwa a no t differen t fro m contro l a t any

samplin g tim e durin g 9 day s o f gasolin e treatmen t o f mal e rata . Post-exposur e deca y o f rena l a ? 

### -globuli nandhyalin e droplet s accumulate d durin g gasolin e exposur e Sinc e mos t exposure s t o fue l hydrocarbon s ar e intermittent , studie s t o
asses s th e effec t o f a variabl e perio do f post-exposur e recover y o nindice s o f gssoIine-induce dnephropsth y wer e conducted . Simila r t o th e studie s already presented , ors l administratio no f gasolin e fo r 9 day s cause da marke dincreas e inth e renal , conten t o f a . -globuli nwhil e th e leve l o f hepati c globuli n protei nwa s no t differen t from control s (Fig.1) . Correlate dmorphologica l studie s showe dths t increase drena l a « -globuli nwa s accompanie db y accumulatio no f hyalin e droplet s i nPC T epithelia l cells , exfoliatio no f cell s int o tubul e lumin a andinfrequen t developmen t o f foc i o f epithelia l cel l proliferatio n(Fig.2) . Analysi s o f rena l tissu e from rat s s1 1 owe d3 dsy s o f
recover y

followin g 9 day s o f gasolin e exposur e showe dtha t Bjy-globuli nlevel s - 7 -P.3 7



### wer e decrease dsbou t 75 % fro m th e highlevel s observe ddurin g exposur e an ddi d
no t diffe r fro a level s i nrat s receivin g salin e onl y (Fig.l) . Afte r recovery fo r 6 o r 9 days , rena l a « -globuli nwa s simila r t o tha t observe da t th e 3 da y
recover y

interval . Examinatio no f rena l tissu e section s fro m rst s allowe d3 day a post-exposur e recover y showe dths t th e numbe r o f hyslin e droplet s wa s reduce dfrom

pea k level s observe dimmediatel y afte r gasolin e treatmen t (Fig.3) . However , th e complet e resolutio no f hyalin e drople t accumulatio nappeare dt o requir e 6 t o 9 day s (Fig.4) . Effec t o f estradio l o nhepati c an drena l a^ , -globuli nan dhyalin e droplet s durin g recover y

from gasolin e treatmen t Treatmen t o f mal e rat s wit hestradio l decrease dth e hepati c conten t o f a„- globuli nb

y 74 % b y post-gasolin e exposur e day 3 compare dt o rat s receivin g gasolin e withou t hormon e treatmen t (Fig.l) . Th e suppressiv e effec t o f estradio l
onhepati c synthesi s o f a . -globuli nincrease db y post-exposur e dsy s 6 and9 , althoug hestradio l treatmen t wa s discontinue do npost-exposur e day 3 . Thus , level s o f a . -globuli ni nth e liver s o f estradiol-treate drat s wer e onl y abou t 3.5%.o f value s observe di nrats allowe dt o recove r fro m gasolin e administratio n withou t hormon e fo r 6 an d9 day s (Fig.l) . Reflectin g th e effect s o f estradio l
o nhepati c synthesi s o f a « -globalin y th e rena l conten t o f thi s protei nwa s decrease db

y 25% , 41 % and52 % o nday s 3 , 6 snd9 post-gasolin e exposur e whe n compare dt o th a kidney s o f rats no t receivin g th e hormon e (Fig.l) . Histologi c analysi s o f kidne y

tissue s fro

m estradiol-treate drat s reveale dtha t hormon e treatmen t als o appeare dt o accelerat e th e rat e o f remova l o f gasoline-induce d hyalin e droplet s suc hthat , compare dt o rat s no t receivin g th e hormone , hyalin e drople t numbe r andsiz e wer e nea r contro l level s a t 3 day s post-exposur e (Fig.5 ) an db

y 6 dsy s o f recovery , rena l PC T wer e no t differen t i nappearanc e fro m ssline-treste dcontrol s (no t shown) .



## -8 -P.3 8

TITIO O 

### o&cStf

|°)Ppoo(n)fe

o to ® ffundoEoato1 no o ©Iboopvatien s i n th o stud yo f gas©lino°5ifc°fc#gc d hyolin o dp@®lo t nophpepath yo f rats

. Thos o aro ; 1 ) th o dio<s@wop

ytlnio t o oongl o odaonootratiiQin ) ©f f «goo©Iin o i o suffffoeiop t t © indue© o3cgira5•{?otsoffaf

e poao l potoatoQ a ©f f s ^ °gfl@fo<a

l on , on^

) 2 ) e 9 

### °gl!@@ial i n on d hyolosa © dp®(°>loto o<§€isaiy

i I ofec^ l <°!iaP0(n)@ goo

© I I Sa o ii(n)fe©no<sofeii®in

) dJoo^Qor

. po(°>odlyoho n goo© I on o o<fciofa3oferofej©f a os topaoaotc d on d th o op®ntono®u s pot o @f poeevop yfro a fjyo B 5a o dp®@l)o

t (nio(°>!n)P©(p>ath

yo o oeeollopoto d feytpoofeson t @f nol o pot o ait h •ootpodi®!

. Synthooo o on d oaepoti® n ©f f e « =§1©k

m io n ©eeup o a t hig h poto s i n ool o pot s (ffciuhau s o t a!. , 19§1)

. Thoo o popoeotop s ap o no t altopo d feyhydp@eapfe© n ospesur o (Gop g © t ol. , 1986 ; 01a@ n o t a I. , X8@<S ; Voa u o t a I. , 5L9H6)

. Fupthopraoro , th o pos@rpti© n ©f f thi o globuli n (p)p®to5 n i o gpoato rtha n 805 3 offffoeion t ami

d ne t i cap

o i po d dupin g hydp©eapb©n°indu<e© d hyolin o dp©g>8of

e nophp@poth y(Voa u o t al. , 198®)

. Thoa o ©fesopvatien o i n conjunction'ait h th o ti£3a-=dopondone o o f e « -globuli n accumulatio n (Tobl o 1 ) sugges t tha t gaselin © caus© s a papi d dirainutie n © f th o p©st°Pos©ppti© n ppocossin g o f e « -globuli n i n

mmWm. 

### rono l PC

To p i th o I i a I colls

. Th o ppoeoo o raoehonossa o f suc h a n offse t i s untxnoo n bu t oa yfeo du o t @ eauoo s in e Du d in g otpuetupa l isodi f ieafeio n © f e „ -glebuli n (KI©s o an d Bus , 19®S) „ onhifeoto@ n ©f f ®P®to@lyto conxyro o activity , altopatoe n o f

m-r 

### felruo ontpalyo®o®cal ) $!. © p altopoto@ n ©f

f pono l onopg ycsotab© ! osffii

. X t ha s feo©n sh®a n PC@csCillj

7 tlhiof e soa°spo< § o ff o e phopcoeekinoti e difforonco s resul t i n ®p@§®[nK°)C^

] rofecsafeiiefii ©f

f 2„2„4°tp5cothyl^onton o (TOP) , a nephrotoxi cgao@lin © eenotituont, , i n th o bidnoy s @f eol o pot s (Chopbonn©a u o t a I. , 1986)

. TOP spcciffieall yoncroooo s hyolin o dp@®to t ff@PQoto@n an d aeeuraulatie n of f e « - glefeulo n i n th o S2 sogaon t ©f f th o pono l pp@aooa l tubulo s o f eaal o rat s (Sfeonard
o t ol. , 1988)

. Rono l lowol s ©f f TOP OOP O GOHSKO I 

### 1 2 h p affto p a singl o ©ra i dos e ©f f TOP an d a t 2 4 on d 4 ® h p OOP O 

### 94 8 an d 45S 3 ©f f souiraa l (Charbonnoa u o t al. , - 9
-

### 1986) . I ncontrast , pea k TM P concentration s i nth e kidney s o f femal e rat s wer e onl y 46 % o f thos e foun di nmale s andwer e attaine d8 h r afte r administratio n b y 2 4 an d4 8 h r post-TM P exposure , level s wer e 53 % an d15% , respectively , o f maximal . Thus , pea k level s o f hydrocarbo nretaine di nth e kidney s o f mal e rat s correspon dtempor e M y to-th e poin t s t whic haccumulatio no f a « -globuli nsnd hyalin e droplet s wer e note di nou r study . I t ma y b e conclude dfrom

thi s tha t
rena l a . -globuli naccumulatio ni s dependen t o ns defec t ina « -globuli n catabolis m

whic hi s associate dwit hpersisten t hig hlevel s o f hydrocarbon . Rapi ddissppearane e o f a . -globuli nfro m th e kidne y

wa s observe dfollowin g terminatio no f gasolin e administratio n(Fig.l) . Th e dat a sho w clearl y

tha t afte r onl y

3 day s o f recover y from gasolin e intoxication , a „ -globuli n accumulate di

nth e kidne y

ha ddissipated . Moreover , th e rena l conten t o f a . - globuli ndi dno t diffe r betwee nrat s allowe d3 , 6 o r 9 day s recover y from gssoline . Thus , i t i s likel y tha t reductio ni

nabnorma l level s o f a , -globuli n
occurre dmor e quickl y tha n3 days . I nspit e o f th e lac k o f specifi c informatio n
o nrena l a . -globuli nconcentratio no nday s 1 and2 post-exposure , i t i s obviou s tha t th e accumulatio no f a « -globuli ncause db y gasolin e exposur e i s a readil y reversibl e phenomenon . Qualitativ e observatio no f th e histologica l appearanc e o f th e kidne y durin g recover y

afte r gasolin e administratio n(Figs. 3

mlmV 

### and4 ) support s thi s conclusio nsinc e hyalin e droplets , fo r whic ha - -globuli n i s s biochemica l marker , ar e greatl y reduce di

nnumbe r an dsiz e b y 3 dsy s o f recovery . Hyalfe e drople t numbe r continue dt o decreas e o nrecover y day s 6 snd 9 ; b y 9 dsy s o f recover y ther e wa s n o apparen t visus i differenc e betwee nkidne y tissu e from

ssIine-treste drat s andthos e allowe d9 day s o f recover y from gssolin e administration . Stonar de t al . (1986 ) observe dtha t th e accumulatio n
o f a 2 

### -globuli ncause db y a singl e dos e o f TM P wa s msxima l 4 8 hour s afte r administration . However , i ntha t study

th e rena l conten t o f ajy-g'obuli n returne dt o pre-exposur e level s onl y afte r seve ndays . Th e difference s i nth e -10 -P.4 0



### rat e o f disappearanc e o f a . -globuli ni nou r stud

y andtha t reporte db y Stonar d
e t al . (1986 ) wit hTU P ma y b e attribute dt o th e fac t tha t TU P i s a poten t nephrotoxi nan dou r study use dwhol e gasolin e wit ha correspondin g decreas e in absolut e TU P dose . Evidenc e ths t hyslin e drople t nephropsthy i nmal e rat a i a a sex-specifi c
even t dependen t o ncontinuou s availabilit y o f hig hlevel s o f a « -globuli nvs s derive dfrom

a nexperimen t o nth e effec t o f estradio l o nrecover y from hydrocarbon-induce dnephropathy (Fig.l) . A s show npreviously , a ~ -globuli n synthe s i s i

nth e I i ve r i s hi ghI y

dependen t o ntestosteron e (Ro y e t a I. , 1975) . Whe nandrogeni c activit y i s decrease db

y castrstio no r administratio no f estrogeni c hormones , a . -globuli nsynthesi s decline s dramaticall y (Ro y e t al. , 1975) . I nou r experiments , subcutaneou s injectio no f estradio l cause da rapid , almos t complet e disappearanc e o f hepati c a . -globuli n(Fig.l) . Althoug hrena l
a - -globuli nlevel s wer e no t a s dramaticall y altere db

y estrsdio l s s thos e o f liver , a significan t decreas e i nth e rena l concentratio no f thi a protei nwa s observe dcompare dbot ht o saline-treate dcontrol s an dt o rat a allowe dt o
recove r from

gasoline-induce dnephrotoxicit y withou t hormon e treatmen t (Fig.l) . Becaus e a . -globuli nresorptio nfro m th e urinar y

ultrafiItrat e i s very efficien t (Via ue t a I. , 1986) , i t wa s no t expecte dths t th e estrsdiol-mediste d alteratio ni

nrena l concentratio no f a - -globuli nwoul db e o f th e sam e magnitud e as " tha t observe dinliver . Th e importan t point , however , i s ths t level s o f tu-globuli ni nth e kidne y

ar e directl y

relate dt o thos e inlive r and tha t th e norm a Ily

hig hcirculatin g level s o f a - -globuli ninmal e rat s mus t b e msintaine di

norde r fo r rena l a . -globuli naccumulatio nt o occu r i nrespons e t o hydrocarbo nintoxication . Thi s poin t i s furthe r supporte db y wor k o f Ksnerv s e t al . (1986 ) an dHobso ne t al . (1986 ) showin g tha t immatur e o r castrate dmal e rat s whic hhsv e n o o r greatl y diminishe da « -globuli nsynthesi s d o no t develop , hyalin e drople t nephropath y whe nexpose dt o hydrocarbon s simila r t o thos e found

-11 -P.4 1



### i nunleade dgasoline . I t i s possibl e tha t th e norma l rat e o f protei ncatabolis m i nrena l lysosomes- , whic hma y b e impaire db

y gasoline , i s restore dafte r gasolin e withdraws l sn daccumulate dproteins , includin g a ~ -globulrn , ar e proteolytics!l y

digested . A

nalternativ e possibilit y i s tha t PC T epitheIi a I
ee l l a containin g accumulate da . -globuli nma y b e irreversibl y

damage dand durin g th e fira t 3 day a followin g gasolin e exposure , thes e cell s sr e the n exfolisted . Althoug heithe r o f thes e mechanism s woul dsccoun t fo r result s show n i nFigur e 1 , the y

hsv e differen t mechanisti c implicstions . Whil e th e firs t possibilit y

woul dsuppor t th e reversibl e nstur e o f fue l hydrocarbon-induce d nephropsthy , th e secon dsuggest s tha t th e rapi donse t o f a nirreversibl e toxicit y

contribute s t o cytotoxicit y andincrease depithelis i cel l replicatio n (Shor t e t al. , 1986) . Furthe r experiment s ar e neede dt o differentiat e betwee n thes e possibilities . Thus , th e dat a presente di nthi s repor t strongl y sugges t ths t gasoline induce da . -globuli nan dhyalin e drople t accumulatio ni nmal e rat s i s rapidl y inducible , i s readil y reversibl e anddependen t o nmaintenanc e o f norma l constitutiv e level s o f circulatin g a . -globulin . Thi s suggest s furthe r tha t significsn t nephrotoxicit y du e t o gasolin e exposur e woul db e likel y t o occu r onl y i nsnima l specie s wit hhigh-leve l proteinuria . Sinc e mal e rat s ar e know n t o exhibi t physiologi c proteinuria , i t i s possibl e ths t thes e snimal s ar e unususll y

susceptibl e t o gssoIine-induce dnephropsthy .

•12 -P.4 2



### ACKNOWLEDGEMEN T

Th e author s ar e gratefu l fo r th e assistanc e o f Scot t Webb , Dr . Kathlee n Smiler , Alexandr a Brady an dLind a L i o f th e Biomedica l Scienc e Department , G M Resesrc hLsboratorie s sndMichae l Mancini , Or . C.V.R . Murt y andOr . Fazlu l Sarka r o f th e Departmen t o f Biologica l Sciences , Oaklan dUniversity . Partia l fundin g o f th e resesrc hequipmen t use dfo r some o f th e experiment s ws s provide d b y NI H gran t AM-14744 .

-13 -P.4 3



### REFERENCE S
Busey , N.M. , sndCockrell , B.Y . (1984) . Non-neoplasti c exposure-relate drena l lesion s i

nrst s followin g inhalatio no f unleade dgasoline . I nRena l Effect s o f Petroleu

m Hydrocsrbons . (M.A . Mehlmsn , CP . Hemstreet , III , J.J . Thorp e sndN.K . Wesver , eds.) , pp . 57-64 . Princeto nScientifi c Publishers , Inc. , Princeton , N.J .
Chsrbonneau , M. , Lock , E.A. , Strasser , J. , andBus , J.S . (1986) . Dispositio no f [ 14 

### C]-trimethylpentan e (TMP ) Pharmacologis t 28 , 17 8 (abstr ) [ C]-trimethylpentan e (TMP ) i nmal e an

dfemal e Fische r 34 4 rats . Th e

Conawsy , CC , Schreiner , C.A. , an dCragg , S.T . (1983) . Mutageni c evaluatio no f petroleu

m hydrocarbons , i

nProceeding s o f th e Symposium : Th e Toxicology o f Petroleu

m Hydrocarbons . (H.N . MacFarland , C.E . Holdsworth , J.A . McGregor , R.W . Cal l an

dM.L . Kane , eds.) , pp . 128-138 . America nPetroleu m Institute , Washington , DC .
Environmenta l Healt hLette r (1985) . Healt hEffect s Institut e repor t o ngasolin e vapors . 2 4 (Oct . 15) , 8 . Gsrg , B.D. , Olson , M.J. , Sarkar , F.H . andRoy , A.K . (1986) . Rena l hyali n drople t snda . -globuli naccumulatio nafte r gasolin e administratio nt o mal e rats . Th e Pharmacologis t 28 , 21 0 (abstr) .
Hslder , CA. , Warne , T.M. , andHatoum , N.S . (1984) . Rena l toxicit y o f gasolin e andrelate dpetroleu m naphtha s i

nmsl e rata . I nRena l Effect s o f Petroleu m Hydrocarbon* , (M.A . Mehlmsn , CP . Hemstreet , III , J.J . Thorp e an dN.K . Wesver , eds.) , pp . 73-88 . Princeto nScientifi c Publishers , Inc. , Princeton , N.J .
Hobson , D.W. , Msttie , D.R. , Bruner , R.H. , Uddin , D.E. , Eurell , T. , an dOlson , C T . (1986) . Effec t o f castratio nonth e developmen t o f 2,3,4- trimethylpentane-induce dhydrocarbo nnephropath y i nth e mal e F-34 4 rat . Th e ToxicoIogis t 6 , 17 3 (abstr) .

•14 -P.4 4

Kanerva , R.L. , Ridder , CM. , Stone , L.C , an dAlden , C.L . (1986) . Characterizstio no f spontaneou s an ddecalin-induce d hyalin e droplet s i n kidney s o f adul t mal e rats . Fd . an dChem . Toxicol , i npress .
Kloss , M.W. , an dBus , J.S . (1985) . Hydrocarbon-mediate d nephrotoxicity . C.I.I.T . Activitie s S , 1-8 .
Loury , D.J. , Smith-Oliver , T. , andB.E . Butterwort h(1986a) . Inductio no f DN A repai r an

dreplicativ e synthesi s (RDS ) followin g exposur e t o unlesde d gasoline . Th e Toxicologist , 6 , 22 7 (abstr) .
Loury , D.J. , Smith-Oliver , T., Strom , . S. , Jirtle , R. , Michslopoulos, G. , sn d Butterworth , B.E . (1986b) . Assessmen t o f unschedule dan dreplicativ e DN

A synthesi s i

nhepatocyte s treate di nvivo an

di nvitr o wit hunleade dgasolin e o r 2,2,4-trimethylpentane . Toxicol . Appl . Pharmacol . 85 , 11-23 .
Lowry , O.H. , Rosebrough , N.J. , Farr , A.L. , an dRandall , R.J . (1951) . Protei n measuremen t wit hth e Foli npheno l reagent . J.Biol . Chem . 193 , 265-275 .
MacFarland , H.N. , Ulrich , C.E. , Holdsworth , C.E. , Kitchen , D.N. , Hslliwell , N.H. , an

dBlum , S.C . (1984) . A chroni c inhalatio nstud y wit hunlesde d gasoline . J . Am . Co l I . Toxicol . 3 , 231-248 .
Neuhaus , O.W. , Flory , W. , Biswas , N. , an dHollerman , C.E . (1981) . Urinar y excretio no f a . -globuli nan dalbumi nb

y adul t mal e rat a followin g treatmen t wit hnephrotoxi c agents . Nephro n28 , 133-140 .
Olson , M.J. , Garg , B.D. , Murty , C.V.R. , an dRoy , A.K . (1986) . Accumulatio no f
a . -globuli ninth e rena l proxima l tubule s o f mal e rat s expose dt o unlesde d gasoline . Toxicol . Appl . Pharmacol. , submitted . Phillips , R.D . andG.F . Egs n(1984 ) Effec t o f C 1 Q - C uisopsraffini c solven t o nkidne y

functio ni

nFische r 34 4 rat s durin g eigh t week s o f inhslation , Toxicol . Appl . Pharmacol . 73 : 500-510 .

-15 -

### Richsrdson , K.A. , Wilmer , J.L. , Smith-Simpson , D. , andSkopek , T.R . (1986) .

Assessmen t o f th e genotoxi c potentia l o f unleade dgssolin e an d2,2,4 - trimethyIpentan e i

nhuma nlymphoblast s i nvitro . Toxicol . Appl . Pharmacol . 82 , 316-322 .
Roy , A.K. , McMinn , D.M. , andBiswas , N.M . (1975) . Estrogeni c inhibitio no f th e hepati c synthesi s o f a - -globuli ni nth e rst . Endocrinolog y 97 , 1501-1508 . Roy , A.K . (1977) . Esrl y event s i

nth e steroids l regulstio no f a - -globuli ni n th e rs t liver . Eur . J . Biochem . 73 , 537-543 .
Short , B.G. , Burnett , V.L. , andSwenberg , J . (1986) . Histopstholog y an dcel l proliferatio ninduce db y 2,2,4-trimethyIpentan e i nth e mal e ra t kidney . Toxicol . Pathol . 14 , 194-203 .
Stonard , M.D. , Phillips , P.G.N. , Foster , J.R. , Simpson , M.G. , sndLock , E.A . (1986) . a - -Globulin : Measuremen t i nra t kidney

followin g administratio no f 2,2,4-trimethyIpentane . Toxicolog y 41 , 161-168 .
Viau , C , Bernard , A. , Gueret , F. , Maldaugue , P. , Gengoux , P. , an dLauwerys , R . (1986) . Iaoparaffini c so l vent-induce dnephrotoxicit y i nth e rst . ToxicoIog y 38 , 227-240 .
Zeiss , L. , Crouch , E.A.C , andWilson , R . (1986) . A possibl e relationshi p betwee ntoxicit y

andcarcinogenicity, . J . Am . Coll . Toxicol . 5 , 137-151 .

•16 -P.4 6



### Tabl e 1
Effec t o f Duratio no f Ora l Dosin g Wit hGssolin e o nRens I sndHepsti c Om -Globuli nConten t

Treatmen t Rena l a - -Globuli nHepati c a « -Globuli n 0*g/m g tota l protein )

Saline , 9 day s 20.6 5 * 0.9 1 1 

### 0.8 0 * 0.1 9 Gasolin e xl d44.1 7 * 4.0 1 "'* * 0.8 2 * 0.1 3

" x3 d66.5 6 * 11.1 3 "»* * 0.7 9 • 0.1 6

• x5 d69.0 4 * 14.1 2 * ' 1.0 8 * 0.0 6

• x7 d82.7 4 * 34.4 0 * * 0.9 7 * 0.1 6

• x9 d63.7 7 * 13.8 6 * * 0.8 4 * 0.0 2

Gssolin e (2. 0 mL/kg ) o r s nequivalen t volum e o f salin e wer e administere db y oavag e onc e dail y

an dtissue s fo r assay wer e obtains ^ 1 8 hour s sfte r dosing , mea n* S.E.M. , n» 3 rat s pe r treatmen t group . differen t fro m saline treate dgroup , p< 0.0 1 (Dunnett' s test) . * * differen t fro m precedin g value , p < 0.0 1 (Duncan' s test) .

•17 -P.4 7



### FICUR E LEGENO S Figur e 1 . Effec t o f recover y time an

destradio l treatmen t o nrena l a ~ -globuN n conten t after terminatio no f gasolin e exposure . Rat a wer e treate d wit hvehicl e alon e (X) , 2. 0 mL/k g gasoline , p.o . fo r nin e day s (s) ) -o r gaaolin e plu s estradio l ( 1 mg/kg , s.e. ) fo r fou r day s beginnin g o n da y nin e o f gaso l ine administration . ( O ) • Dat a ar e expresse da s mea n * SE M a . -globuli nconten t i nlive r andkidney . Arro w indicate s terminstTo no f gasolin e treatmen t o f rata . Significantl y differen t from al l othe r grou pmeans , P < 0.0 1 (Duncan' s test) . Differen t from correspondin g valu e fo r grou pno t receivin g estradiol , P < 0.05 , P < 0.0 1 (Duncan' s test) . Figur e 2 . Photomicrogrsph s o f rens i tissue section s fro m ssline-treate d(2. 0 mL/kg , p.o. , 9 days ; Pane l A ) andgasoline-treste d(2. 0 mL/kg , p.o. , 9 days ; Pane l B ) rats . Enhance dhyalin e drople t (<4^» ) accumulation , correlate dt o a nincreas e i na . -globuli ncontent , i s obviou s i nth e kidne y o f th e gasoline-treate d"animal . Mallory' s Heidenhai nstain . 50X . Figure 3 . Photomicrograp ho f kidne y tissue fro

m a gasoIine-treste d(2. 0 mL/kg , p.o. , 9 days ) ra t allowe d3 day s post-exposur e recovery . Compar e t o Fig. 2 fo r effec t o f recover y tim e o nresolutio no f hysiin e drople t nephropathy . Mallory' s stain . 50X . Figur e 4 . Effec t o f 9 day s post-gasolin e exposur e recover y o nhysiin e drople t (^• ) numbe r i

nrena l PC T epithelia l cells . M s I lory' s stain . 50X . Figure 5 . Acceleratio no f recover y fro m gasoline-induce d(2. 0 mL/kg , p.o. , 9 days ) hyalin e drople t nephropathy b y estradio l treatment . Estradio l ( 1 mg/kg , s.e. ) wa s administere ddail y fo r 4 day s beginnin g on.th e nint hds y

o f gasolin e treatment . Rena l tissu e ws s collecte do nth e fina l da y

o f estradio l treatmen t (thu s s i lowing 3 day s recover y fro m gasoline) . Compar e t o Figs . 2 an d3 t o judg e estrsdio l effect . M s I lory' s stsin . 50X .

•18 -P.48

_P.4 9P.5 1



### 0 9 

## TIM E (DAYS )



# * S 'P.5 2



### GMR-678 5

IMMUNOELECTRON

MICROSCOPI C LOCALIZATION O F ALPHA-2U-GL0BULI N I N TH B MAL E RA T KIDNE YAFTE ROASOLIN E TREATMEN T

B . D . Garg * M . J . Olson * L . C . Li * M . A . Mancini* * A . K . Roy* * •Biomedica l Scienc e Departmen t Genera l Motor s Researc hLaboratorie s Warren , M I 48090-905 8 ••Departmen t o f Biologica l Science s OaklandUniversit y Rochester , M I 4806 3

Fo r Presentatio na t th e Electro nMicroscopy

Socist y

o f Americ a Meetin g Baltimore , Maryland Augus t 2-7 , 198 7P.5 3



### IMMUNOELBCTRO

N MICROSCOPI C LOCALIZATIO N O F o^-GLOBULI N

IN THB MAL E RA

T KIDNE Y AFTE RGASOLIN

E TREATMEN

T
B.D . Garg* , M.J . Olson* , L . Li* , M.A . Mancini* * aa d A.K . Roy* * Biomedica l Scienc e Department , G M Researc

hLaboratories , Warren , M I 4809 0 * Department o f Biologica l Sciences , Oaklan d University , Rochester , M I 4806 3 • •


### Exposur e o f mal e rat a t o gasolin e an d other volatil e hydrocarbon s cause s acut e nephrotoxicit

y accompanie

db y accumulatio

no f hyali ndroplet s (HD ) i n epithelia l cell s o f th e proxima l convolute d tubule s (PCT ) . f e hare show n tha t HD accumulate

dafter gasolin

e intoxicatio

nar e partiall y

compose

d o f a « - globuli n' , a protei nfoun d exclusively

i nadul t mal e rat e . Ultrastructura l examinatio

no f PC T durin

g gasolin

e intoxicatio

nreveale

d HD t o b

e enlarge

d phagolysosome s . I t i s hypothesise d tha t metabolite s o f gasolin e hydrocarbon s bin d rerersibl y

t o a „ -globulin , perhap s limitin g lysosoma l biodegradatio nof thi s protei n. a * -Globuli naccumulatio nma y als o occu

r due t o mechanism

s suc h a s hydrocarbon-mediate dinhibitio

nof lysosoma l proteolyti c ensym s activity . Ultrastructura l examinatio nof PC T durin

g gasolin

e intoxicatio

nreveale

dH D t o b e enlarge

dphagolysosome s Thes e studis s wer e conducte d t o determin

e th e distributio

no f a « -globuli nwithi nepithelia l cell s of PC T durin g gasolin

e intoxication . Mal e F-34 4 rat s wer e administere d unleade

d gasolin

e or 0.9 % NaC l solutio n(2. 0 •L/Kg , p.o. , onc e daily , nin e days ) an

d 2 a m slics s o f saline-perfuse dkidne y wer e fixe

d b y immersio

nin2.5 % glutaraldshyde , 0. 1 M sodiu m cacodylat e (p

H 7.4) . For iamunolocalisatio nof a « -globulin , thi nsection s onuncoate dnickl e grid s wer e incubate dwit hsaturate

d sodiu

m metaperiodate , washed sequentially indeionise

d wate r an

dDulbecco' s phosphat e buffere d salin e (DPBS) , the

nplace d in3 % noraa l goa t serum/0.I Xbovin e seru m albumin/DPBS . Then , polyclona l rabbi t Ig Gagains t ra t a „ -globuli n(1-6 0 Ig/aL ) wa s applia d t o th s sections . Afte r washin g inDPBS , section s wer e treate d wit hcolloida l gold-conjugate dgoa t anti-rabbi t Ig S for 3 0 min , washed inDPB S an d deionise

d wate r prior t o urany l acetate/lea

dcitrat e atainin

g an d electro

nmicroscopy .
I nsalins-trsate

d rats , a fa

w phagolysosome s ( 2 /t m diam. ) o f unifor

m sis s with roun dor ora l contour s wer e pressnt inths cytoplas m of PC T spithslia l cell s (Fig . 1) . Ther e wa s a narked increas e i nth e sis e ( 9 ftm) an d number o f phagolysosome s i nepithelia l cell s of th e PC T followin

g treatment o f rat s with unlsade d gasoline . Man

y o f th s enlarge

d phagolysosome s wer e angula r in sectiona l profil e an dfrequently

containe

d electron-dens e crystalloi dbodie s (Fig . 2) . Ther e wer e n o othe r alteration s visibl s i nsubcellular organelle s withths exceptio

no f a fe

w cell s showin

g araa s of cytoplasmi c clearing . Th e imaunogol d technique specifi c fo r a « -globuli nshowe d exclusiv

e localisatio

nofP.5 4



### thi s protei ninth e matri xo f phagolysosomes ; n o a « -globuli nwa s observe d outsid e th e phagolyaosoaes . Smalle r phagolysosome s possesse d unifor

m distributiono f au -globuli nwithi nthsi r matrices . Large irregula r phagolysosoass , however , ha d deposit s o f a * -globuli nlocalise dalmos t exclusivel y

ore r th

e electron-dens e crystallin e inclusions , suggestin g a n aggregatio

no f a 2 u 

### "8l°^ u 

### ^ n

### * a 

### thes e structure s (Figs . 3,4) . Phagolysosome s (secondar y lysosoass ) ar s forme d b y associatio

no f sndocytoti c vacuole s containin g engulfe

d proteinaceou s materia l froa th e urinar

y ultrafiltrat e wit hprimar y lysosoass . Th

e iaaunogol d techniqu e showe

d prsferentia l localisatio no f a . -globuli nwithi nphagolysosomes . Furthermore , a s electron-dens e crystalloi d phagolysosome ! inclusion s formed , a „ -globuli n becam e localise

donl y withi nthes e condense d area s o f th

e matrix . Thus , i t i s conclude

dtha t a defect , induce d b y gasoline , of rena l lysosoma l proteolysi s result s insequestratio no f large amount s o f o u -globuli nincell s o f th e PCT . Furthermore , a « -globuli nform s a major component o f th e intralysosoaa l crystallin

e inclusion s characteristi c o f hyalin e drople t nephropath y cause db y hydrocarbons . Reference s
1 . CA . Haide r e t al. , Rena l Effect e of Petroleu a Hydrocarbons , Princeton , NJ : Princeto

nScientifi c Publishers , Inc . (1984 ) 73 . 2 . I.M . Buse

y an dB.Y . Cockrell , op . cit. , (1984 ) 57 . 3 . M.J . Olso

ne t al. , Toxicol . Appl . Pharmacol . (1987 ) inpress . 4 . B.D . Gar g e t al. , The Pharaacologis t (1986 ) 28 , 210 . 5 . A.K . Roy , Burop . J . Biochea . (1977 ) 73 , 537 . 6 . M . Charbonnea

uet al. , Th

e Toxicologia t (1987 ) 7 , 89 . Figur e Legend s FIG.l . —Bpithslia l cal l o f PC T froa a contro l ra t showin g roun d or ora l cross-sectionedphagolyaosoae s (-*>•) . x2,40 0 FIG.2 . —Th s Euabe r an dsis * o f phagolysosome s wa s markedl y increase

db y treatmen t wit hgasoline . Man y o f th

e organelle s hav

e assume

d rectangula r an

dirregular shape s aa d contai nelectron-dens e crystalline core s (- • ) . FIG.3 . —Tw o large phagolysosome s are labele d withgold-tagge

d antibod

y t o tt. -globuli n(punctat e electron-dens e material ) demonstratin g th s presenc e o f thi s protei ni nth o phagolysosomes . Not e absenc e jj| : extralyaosoma l labeling . x2,40 0 . ' • - " FIG.4 . —Highe r magnificatio no f phagolysosome s showin g electron-dens e antigen-antibod

y complexe s labelin

g a^-globulin . x2,40 0P.5 5P.5 6

^1 » " W * a-iS* " *3< L . mm .'P.5 7



# GMR-5597

.*?•••• • 

# HYAUN E DROPLE TAN D 0£ 2u 

# . -GLOBULINACCUMU LATIO N ( N W E KIDNEY S O F MALE RATS : AFTE R GASOUN E ADMINISTRATIO N

AV ' 

# M . J . Oteor v B . D.Gaf g Departmen t

^•i^r^'^'-'r f 

### :..-.*.. ' 

# Octobe r 27,198 6



# • lesearc h L ' Warren . Michiga n 4809 0

MA YB E OlSTRlBUTE O OUTSID E GENERA L MOTOR S . No t t o be Reproduce d Withou t Permissio nP.5 8



### GMR-559 7

HYALIN E DROPLE T AND a 2u 

### -GL0BULI N ACCUMULATIO N

I N TH E KIDNEY S O F MAL E RAT S AFTE RGASOLIN E ADMINISTRATIO N

M.J . Olso nan

dB.D . Gar g Biomedica l Scienc e Departmen t Genera l Motor s Researc hLaboratorie s

Warren , M I 4809 0

F.H . Sarka r an

dA.K . Ro

y Biologica l Science s Departmen t OaklandUniversit y Rochester , M I 4806 3

Presente dOctobe r 28 , 198 6 a t th e 16t hConferenc e onToxicology , Dayton , Ohi oABSTRAC T

Saturate dbranched-chai naliphati c hydrocarbons , foun di nmoto r fuels , induc e a uniqu e nephrotoxicit y i nmal e rats . Unleade dgasolin e administere dt o male rat s (0.0 4 - 2. 0 mL/k g bod y weight , p.o. ) fo r nin e day s cause da marke dincreas e i n th ©numbe r an

dsize o f hyalin e (protei nresorption ) droplet s i nepithelia l cell s o f th e proxima l convolute dtubule s (PCT ) an d increase dcellula r exfoliatio na t highdose levels . No othe r treatments ©I ate dpathologica l effect s were observe di nth e glomeruli , dista l tubule s o r medulla . Th e rena l conten t o f ts

« -globulin , a protei nsynthesize di nth e live r an

dexcrete di

nthe urin e o f mal e rats , wa s determine db y radioimmunoassay t o b e increase di na dose-relate dmanner ; maxima l accumulatio nwa s 440 % o f control . Subsequently , we observe dtha t bot hhyalin e droplet s an de „ -globuli nwere increase d significantl y

afte r a singl e dos e o f gasolin e (2. 0 mL/kg,p.o.) . Immunoperoxi das e stainin g o f kidney tissu e fo r a « -globuli nreveale dincrease dprotei n conten t i

ngasoline-treate d rat s an dTarg e accumulation s o f antige nwer e localize d i

nsome PC T epithelia l cell s whic hcontaine dhyalin e droplets . Th e hepati c concentratio no f a „ -globuli nan dthe quantit y

o f a „ -globuli nmRN A i n live r wer e no t altere d b y gasoline . Accumulatio no f a. , -globuli nwa s reverse d withi nthre e day s o f terminatin g gasolin e administration . A hydrocarbon-induce d defec t i

nthe rena l lysosoma l degradatio no f lo w molecula r weigh t proteins , rathe r tha nincrease dsynthesi s o r defectiv e metabolis m o f a « -globulin , appear s t o cause hyalin e drople t accumulation .

INTRODUCTION

Some petroleu

m hydrocarbo nmixture s use da s moto r fuels , solvents , pain t an d varnis hadditives , degreasin g agent s an dindustria l intermediate s caus e dose dependen t alteration s i nth e for

m an dfunctio no f th e rena l corte xfollowin g subacut e o r chroni c exposure (1) . Th e importanc e o f understandin g hydrocarbon mediate dnephropath y increase dgreatl y followin g th e observatio ntha t rat s expose dfo r 1 8 o r mor e month s t o wholl y volatilize d unleade dgasoline exhibite d a nincrease dincidenc e o f rena l carcinom a (2) . Th e occurrenc e o f tumor s wa s sex - an

dspecies-specific ; mal e rat s onl y exhibite dtreatment-relate d rena l neoplasia . Analysi s o f gasolin e an dsome o f it s nephrotoxi c constituen t hydrocarbon s i

nshort-term

assay s fo r genotoxicit y ha s produce dequivoca l o r negativ e result s (3) . Thus , researc hha s bee ndirecte dtowar delucidatin g th e mechanism s b

y whic hhydrocarbon s induc e over t nephrotoxicit y i nthe hop e tha t a nepigeneti c even t relate dt o toxicity , suc ha s anincrease drat e o f restorativ e cel l replication , ma y explai nthe occurrence o f rena l tumors . Th e primar y

site o f toxicit y o f hydrocarbo nmixture s simila r t o gasoline , suc ha s je t fuels , C 1Q -C„

_isoparaffini c solven t o r Stoddar dsolven t appear s t o b e epithelia l cell s o V tii e rena l PC T and , b y ligh t microscopi c examination , present s a s anaccumulatio no f protei nresorptio ndroplet s (1) . Althoug ha constitutiv e populatio no f droplet s exists , overabundanc e o f thes e droplets , als o know

na s hyalin e droplets , i s interprete da s a pathologica l alteration . Recently , i t wa s show ntha t th e rena l conten t o f a „ -globulin , a majo r urinary protei nfoundexclusivel y i nmatur e mal e rat s \*)» wa s increase dmany-fol d durin g exposure t o eithe r saturate disoparaffini c solven t o r decahydro naphthalen e (decalin) . a„ -Globuli nha s bee nlinked inferentiall y t o the developmen t o f hyaline drople t nephropath y sinc e volatil e hydrocarbon-induce d nephropath

y i s limited t o male rats . Species , whic hlac k a

_-globuli n(mice , -2 -P.6 0

dogs , monkeys) , sho w n o evidenc e o f hyalin e drople t accumulatio ni neithe r sex' . Th e studie s describe dher e wer e performe dt o characteriz e histologica l an d biochemica l change s i nth e kidney s o f mal e rat s afte r short-term treatmen t wit h unleade dgasoline . Ora l administratio nwa s usedi nanattemp t t o develo pa cos t an dtime-efficien t metho dfo r inducing th e earl y change s characteristi c o f hydrocarbon-induce d nephropathy . Specifi c analysi s o f th ©kidney s wa s performe d to.examin e whethe r a . -globuli nwa s accumulate ddurin g gasoline intoxicatio n an dt o determin e th e subcellula r dispositio no f thi s protei nwithi nth e kidney .

MATERIAL S AN

D METHOD S
Experimenta l animal s an dchemical s Mal e Fische r 34 4 rat s (CD F (F-344)/Cr I BR ; Charle s Rive r Laboratories, Wilmington , MA ) weighin g approximatel y 25 0 g wer e usedthroughou t thes e studies . Rat s wer e administere dphysiologica l salin e o r a commerciall y availabl e unleade dgasolin e b y intragastri c intubation . I nsome experiments, animal s were allowe dt o recove r fo r a variabl e perio dafte r terminatio no f gasolin e administration . Histologica l procedure s Rat s were anesthetize dwit hpentobarbital , perfuse dvi a th e hear t wit hcol d 0.9% sodiu m chloride an

da complet e necrops y wa s performed . Kidney s an dliver s wer e removed , fixe db y immersioni

n10 % neutra l buffere dformalin , processe d routinel y

an dembedde di

nparaffin . Tissue s fo r immuno-localizatio no f a „ - globuli nwere fixe db y immersioni

n3.6 % formalin , 0. 2 M mercuri c chlorid e fpH 7.4 ) the nprocesse dan dembedde di

nparaffin . Seria l section s o f kidney wer e staine dwit hMallory' s Heidenhai nmetho do r immunostaine d fo r a „ -globuli nwit hth e avidin-bioti nprocedur e (5) . Briefly , th e tissu e wa s deparaffinized , rehydrate dan dincubate di

na solutio no f 3 % norma l goa t serum , 3 % bovin e seru m an d2% gelatin . Section s wer e the nincubate d wit hpolyclona l anti=e „ -globuli n(rabbi t IgG ) fo r 3 0 mi nan dwashe dwit h phosphat e buffere dsaline (PBS ; 0.1 4 M NaCl , 0.0 1 M

Na-phosphate , pH 7.4) . The section s were furthe r incubate dfo r 3 0 mi nwit hbiotinylate danti-rabbi t Ig G (goa t IgG ) an

dwashed . A

fina l 3 0 mi nincubatio nwit hfreshl y prepare davidin bioti nreagen t wa s followe db y extensiv e washin g i nPBS . Th e tissue s wer e the n place di

n0.0 5 M

Tris-HCI , pH 7.0 , containin g 0.001 4 U 3,3'-diaminobenzidin e tetrahydrochlorid©an d0.00 3 M

hydroge nperoxid e fo r 1 5 min . Th e reactio nwa s terminate dwit ha 1 5 mi nrins e i nrunnin g wate r an dth e section s wer e counterstaine d wit hHarri s hematoxylin . Dot-blo t assay

o f c ? -globuli nmRN A i nlive r

- Tota l hepati c RNA , extracte dwit hpheno l an dsodiu

m dodecy l sulfat e wa s used a s th e startin g materia l t o isolat e pol y (A)-containin g RN A b y oligo(dt) - cellulos e chromatography . Seriall y dilute d poly(M-containin g RN A wa s spotte d onnitro-cellulos e filter s an dhybridize dwit hP-labele d a « -globuli ncDN A (6) . Afte r hybridization , quantitativ e bindin g o f th e prob e wa s determine db y autoradiograph

y an dsoft-lase r scannin g densitometry .
-3 -

ineiiat i M^atMamM iP.6 1



### Radioimmunoassa y

o f a, , -globuli ninkidne y andlive r Homogenate s o f whol e kidne y andlive r wer e prepare dandcentrifuge da t 100,00 0 xg ; th e supernatan t flui dwa s use dfo r doubl e antibod y RI A o f a „ - globuli na s describe db y Ro y (7) . Th e concentratio no f a « -globuli ni ntH e supernatan t fractio nwa s expresse da s fig a „ -globuli npe r mg tota l protein .



## -4 -RESULT S

Figur e 1 . Seria l section s o f rena l cortica l tissu e fro m a saline-treate d ra t (2. 0 mL/kg , p.o. , 9 days ) prepare dwit hMallory' s Heidenhai nstai nt o demonstrat e hyalin e droplet s (Pane l A ; • • ) o r th e immuno-peroxidas e metho dfo r localizatio no f a « -globuli n(Pane l B;^-) . Not e degre e o f correlatio nbetwee narea s whic har e positiv e fo r bot hstains . 50X . Figur e 2 . Rena l seria l section s fro m a gasoline-treate d ra t (2. 0 mL/kg , p.o. , 9 days) . A

nincreas e i

nhyalin e drople t numbe r an dsize i s eviden t i n compariso nt o Fig . 1 . Compar e th e localizatio no f Mallory' s stain positiv e hyalin e droplet s (Pane l A ; • • ) wit harea s whic hstai n intensel y

fo r a - -globuli n(Pane l B;^») . 5 0 X . Figur e 3 . Accumulatio no f a „ -globuli ni nth e kidney

a s a functio no f gasolin e dose . Gasolin e o r cor noi l vehicl e wer e administere d b y gavage once dail y fo r nin e consecutiv e days . 1 8 h r afte r th e fina l administratio n o f gasoline , kidney (*f) ) an

dlive r (O ) tissue s were sample dfo r RI A determinatio no f a „ -globulin . Dat a ar e plotte da s mea n* SD a „ - globuli ncontent , n= 3 - 7 rat s pe r treatmen t group . p< 0.0 1
• • compare dt o contro l (Dunnett' s test) , p< 0.0 1 compare dt o nex t lowes t dose (Duncans' s test) . Figur e 4 . Dot-blo t hybridizatio no f hepati c mRN A wit hcDN

A fo r a „ -globulin . Lan e A ; pol y

(A)-containin g RN A fro m a saline-treate d rat , lane B ; RN A fro m a ra t treate dwit h2. 0 mL/k g gasoline , p.o . fo r 9 days . Row 1 a 1. 0 /i

g RNA , row s 2- 4 = seria l 1: 1 dilution s o f RN A fro m ro w 1 . Origina l densitometr y trace s o f lane s A an dB ar e shown . B/ A i s th e rati o o f area s unde r th e curv e fo r correspondin g dot s i nlane s B an

d A .

• 5

-P.6 3



### * / - - .* . •

. 

## .*£

* - , * a

a t

* m > • 

# 1

^3 >
Figur e 1 - 6

-P.6 4



# ^T1>mv . ktmWmMr. 7r > raMO T r 

# k.-..;\ ;'..• . •"/ ' • 7 4

• m ^

if



### • 

### tm~T e >

Figur e 2 - 7 -

### 15 0

4 x 10" 4 4 x 10- 3 4 x 10- 2 4 x 10" 1 

## Gasolin e Dos

e (mL/kg )

Figur e 3 - 8 -

### 1 2 3 4
A B

IOO ,

0' lOO i

B/ A (% ) n o * i i n 10 1



### Figur e 4 - 9 -

### Tabl e 1

Tim e Cours e o f Rena l an dHepati c a « -Globuli nAccumulatio nDurin g Ora l Dosin g Wit KGasolin e

Treatmen t Rena l a „ -Globuli nHepati c a „ -Globuli n (jig/mg tota l protein )

Salin e x9 d20. 7 * 0. 9 l 0.8*0. 2 Gasoline xl d44. 2 * 4. 0 **'* * 0. 8 * 0. 1

" x3 d66. 6 * 11. 1 **'* * 0. 8 * 0. 2

" x5 d69. 0 * 14. 1 * * 1. 1 * 0. 1

" x7 d82. 7 * 34. 4 * * 1. 0 * 0. 2

" x9 d 63. 8 * 13. 9 * * 0. 8 * 0. 0

mea n* S.E.M. , n= 3 rat s pe r treatmen t group . differen t fro m saline treate dgroup , p< 0.0 1 (Dunnett' s test) . differen t fro m precedin g value , p< 0.0 1 (Duncan' s test) . Gasoline (2. 0 mL/kg ) o r a nequivalen t volume o f saline were administere db y gavag e once daily .

-10 -P.6 8



### Tabl e 2

Effec t o f Recover y Tim e onRena l a „ -Globuli nConten t Afte r Ora l Gasolin e Administratio n

* * Treatmen t Rena l a „ -Globuli n 2 u (/ig/m g tota l protein )

Saline , 2. 0 mL/k g x9 d24. 2 * 4. 2 X(3 ) Gasoline , 2. 0 mL/k g x9 d127. 0 * 11. 0 (6 ) Gasoline x9 d• 3 d recovery 33. 0 * 5. 1 (3 ) Gasolin e x9 d• 6 drecovery 31. 9 (2 ) Gasoline x9 d • 9 drecovery 26. 2 * 4. 1 (3 ) Gasoline x9 d • 1 2 d recovery 34. 0 * 5. 2 (3 )

mea n* S.E.M. , ni nparentheses . differen t fro m al l othe r grou pmeans , p< 0.0 1 (Duncan' s test) . Rat s wer e administere dsalin e o r 2. 0 mL/k g gasoline b y gavage once dail y fo r nin e consecutiv e day s followe db y th e recovery

period s shown .

•11 -P.6 9



### SUMMAR YAN D CONCLUSION S

W e hav e demonstrate dtha t short-ter m ora l administratio no f unleade d gasolin e t o mal e rat s cause s hyalin e drople t nephropathy , th e characteristi c initia l rena l lesio nobserve ddurin g chroni c inhalatio no f wholl y volatilize d gasolin e b

y rats . Microscopicall y th e rena l patholog y wa s confine dt o th e PC T an dwa s characterize db y accumulatio no f hyalin e (protei nresorption ) droplets . Morphologica l an dbiochemica l characterizatio no f th e kidney s o f gasoline intoxicate drat s showe dthat : 1 . Ora l administratio no f unleade dgasolin e cause dsimultaneou s increase s in th e numbe r o f rena l hyalin e droplet s an da

_-globulin ; dose s a s smal l a s 0.0 4 mL/k g induce dsignifican t rena l accumulatio no f a „ -globulin . 2 . Hyalin e drople t nephropath y wa s induce dwithi non e da y o f initiatin g ora l administratio no f unleade dgasoline ; thre e day s o f treatmen t induce dmaxima l accumulatio no f a „ -globuli nan dhyalin e droplets . 3 . Localizatio no f a „ -globulin , b y a specifi c immunochemica l reaction , wa s demonstrate dinPC T whic hwer e mos t severel y involve dingasoline-induce d hyaIin e drople t accumulation . However , no t al l Mallory' s stain-positiv e hyalin e droplet s wer e recognize da s containin g <x « --globulin . 4 . Th e hepati c conten t o f a

_-globuli nan dexpressio no f it s specifi c mRNA in th e live r wer e no t significantl y affecte db

y gasolin e administratio na t time s whe nrena l a . -globuli nwa s increased . 5 . Hyalin e drople t nephropath y andth e rena l accumulatio no f a « -globuli n induce db

y gasolin e wer e rapidl y reversibl e afte r subacut e administratio no f gasoline . Experiment s ar e inprogres s t o defin e th e sequenc e o f event s precedin g accumulatio no f a « -globuli ni nth e kidney . Th e goa l o f thes e experiment s i s t o reac ha mechanisti c explanatio nfo r hydrocarbon-induce dhyalin e drople t nephropathy .

•12 -P.7 0



### REFERENCE S

1 . Rena l Effect s o f Petroleu m Hydrocarbons , M.A . Mehlman , G.P . Hemstreet , III , J.J . Thorp e andN.K . Weaver , eds. , Princeto nScientifi c Publishers , Princeton , NJ , 1984 . 2 . MacFarland , H.N. , C.E . Ulrich , C.E . Holdsworth , D.N . Kitchen , N.H . Ha 11iweI I an

dS.C . Blum , J . Am . Coll . Toxicol . 3 : 231-248 , 1984 . 3 . Loury , D.J. , T . Smith-Oliver , S . Strom, R . Jirtle , G . Michalopoulo s an d B.E . Butterworth , Toxicol . Appl . Pharmacol . 85 : 11-23 , 1986 . 4 . Roy, A.K . andO.W . Neuhaus , Proc . Soc . Exp . Biol . Med . 1 : 894-899 , 1984 . 5 . Hsu , S.M. , L . Rain e an dH . Fanger , Am . J . Clin . Path . 75 : 734-738 , 1981 . 6 . Roy, A.K. , B . Chatterjee , W.F . Demyan , T.S . Nat han dN.M . Motwani , J . Biol . Chem . 257 : 7834-7838 , 1982 . 7 . Roy, A.K. , Eur . J . Biochem . 73 : 537-543 , 1977 .

-13 -P.7 1



### UNLEADE D GASOLINE-INDUCE D PROTEI NDROPLE T NEPHROPATHY : RELEVANC E O F AMAL E RA T SPECIFI C SYNDROME T O HUMAN S

Miche l Charbonneau , Bria nG . Short , Edwar

d A . Loc k an d Jame s A . Swenber

g Chemica l Industr

y Institut e o f Toxicolog y Department o f Biochemica l Toxicolog y an d Pathobiolog

y P . 0 . Bo

x12137 , Researc

hTriangl e Par

k NorthCarolin

a 2770 9

Tn * Auoelollo n Dmdlcolm d t o Al> Pollutio n Contro l an d Moiardou t Wotl m Manogmmmn t 

## Fo r Presentatio n at th e 80t h Annua l Meetin g o fAPC A Ne wYork , NewYor k Jun e 21-26,198 7

### represent s apossibl e oourc o o f huma n asposur o vi ath e consumptio no f drinkin g wate r

containin g water-oolubl o chemical s fro mUG

. RENA LEFFECTS O

FUNLEADE

D GASOLIN

EIN ANIMAL

S U Gcarcinogenicit y i naaisal o wa o toote di n a2-yoa r otudy { osposur e t o vapor o o f

O Gcause da aineroaoo diaeidoac Go f roaa l adaaoaa s an d eareiaoma o i a©al o FischGr-34 4 rato , bu t ao t i afemal e rat o @ £oitha r

se x o f mic e (2,3)

. Subchroai e inhalatie aGzspeour o t o U Gvapor o produce da ©al e rat-spacifi e nephropath y characterise db y a aiaeroaoo dprotei n (hyaline ) dropla t

fersatie ai aopitholia l coll o o f th o prosisa l tubules , wit haccompanyin g degenerativ e change s o f th o proxima l tubul e i ath e rena l cortea , aa dtubula r dilatatio n an deaot o a t th e juactioa s o f

th o iane r

an d oute r otrip o o f

th o oute r

©odulla(4) o Evaluatio n o f

th o aophrotoxi e preportio o © f ocvora l eo©penoat o o f U G do©oaotrato dthat , a o aelaao , braaeho daliphati c alkaao o (iooparaffiao ) ar o primaril y raoponoibl o fo r th o aophrotoxi e actio a© f U G(4)

. Among th o aophrotesi e hydroearboa o typicall y foua di aOG 0 

### 2 9 

### 2 9 

### 4°tei@othylpQataa e (TKF) D 

### a choaica l adde da s a aoati-kaoekia g agoat , wo o ©booste dt © b o ver y aetiv q(5)

. Thorafera , mW hao boo auoo do o apretetyp o compoun dt o otud y t h e aechanio © o f

OG=iaduee daephreteaieit y i arat o (5,7,8,9,10) 0 Aooesomea t

o f th e geaotosi c propertie s o f ualeade dgasolin e b y a batter y o f

test s ha s show n tha t O Gi o aaoa-genotoxi c chemica l (11,12,13)

. X t ha s bee npostulate d( 9,14 ) tha t tumo r productio n i o cause db y a chroni c regenerativ e proceoo . Lour y £ t a^
. (13 ) reporte dtha t ©al e rat s trGato d(inhalatio no r gavage ) wit hU

Gohowe da©arko diaeroao o i ath o perceatag o o f

kidne y cell o i aS-phaoG , i.o

. coll o undergoin g DN A syntheoio , whoraa o col l proliforatio n wa o ao t oigaificaatl y enhance di n femal e rato
. Doin g a©ulti-otag o careinogeneoi o modo l (15,IS,17 ) i t wa s propooo dtha t

th o productio n o f ©aligaan t

tumor s rosult o fro © repeate d geneti c change o i ath o cell o o f th e hoo t

tiooue

. An adaptatio n o f thi s mode l fo r

aoa-gaaotosi c chemical o producin g tumor o afte r chroni c administratio nt o aaimal o coul dpootulat o that s 1 ° Cello , i awhic h spoataaoou s DN

A altGration o naturall y occur , replicat e followin g chemically-iaduce dcoll.proliferatio acauoia g amutate dcoll

. 2

- Th e mutate dcel l uadergoe o clona l expaaoio adu e t o iacrease dcel l proliferation

. 3 ° Thi s greatl y increase s th e likelihoo do f asecon d spontaneou s mutatio n occurring , leadin g a n increase dincidenc e o f rena l cancer . CIIT' aapproac ht o stud y UG-iaduce drena l tumor s ha s therefor e bee n focusec^o n UG-induce dcol l proliferation

.

ONLEADE D GASOLINE-INDUCE

D CEL LPROLIFERATIO

N A 21-da y hiotoautoradiographi c stud y wa s undertake n t o asses s th e intra-rena l localisatio n o f UG-induce dcel l proliferation

. Thre e mal e Fischer-34 4 rat o pe r grou p wer e expose dt o 0 , 2 , 20 , 20 0 o r 200 0 pp mU

G ( 6 hr/day , 5 days/wee k fo r 3 wooko)

. Oomoti c pump s fillo dwit h[ H]

- thymidin e ( H-TdR ) wer e implante doubcutaneouol y o n da y 1 2 t o aoour e a constan t

releao e o f

H-TdRdurin g th e las t wee k o f oxpooura . Th o animal s wer e kille d1 da y afte r th e las t

exposure , an dperfuoioa-fiso dkidney s wer e remove dan dprocesoo dfo r ligh t an ddar k

fiol dautoradiographi c examinatio n o f

rena l cel l proliferation

. Percentage s o f labolo dcoll s (S

phase ) ove r

tota l cell s wer e determine dfo r th e Pl , P 2 an dP 3 segment s o f th e proximal , tubule

. Figur e 1 show s tha t th e percentag e o f labelo dcell s ove r tota l cell o wa s significantl y ( p < 0.05 ) increase dfo r th o P 2 segmen t - 2 -

### rena l DN A measure di n kidney s o f femal e Fiecher-34 4 rat s treate dwit h50 0 mg/k g TM

P4 o s 6 day o previousl y wer e no t significantl y differen t fro m contro l value

. Therefore , TMP-induce drGna l DN A synthesi s i s incroaoe di n adose-dependen t

manne r afte r th e administratio n o f aoingl e ora l dos e o f TM Pt o mal e bu t

no t femal e rato
. PROTEI N DROPLE

T ACCUMULATIO

N Th GP 2 oogaea t

corrospoad o t © th o rGgie ao f th o aophro awhor o th e lo w ©oloeula r

weigh t

protoiao , filtoro diat o th o tubul o luso aa t th e gle©oruluo , ar e reabsorbe db y ondoeyteoi o iat © coll o © f th o proxima l tubtslo o (18 ) o ' Th o oadeeytetie

. vooielo o (oadoooaeo ) fuo o wit hlyoeoomes , aa dth e protoia o coataiae di ath o rooultia g ondolyoooeme s ar e hydrolyse d t o .thoi r

constituen t

aaia o acid o b y lyo©o©ma l Gagyae o (19)

. @2u=Gl©bulin , alo w ©oloeula r

woigh t protei aprodueo db y ©al o bu t ae t fosal o rat o i s roaboerbo db y thi o precoo o (20)

. Haldo r o ^ al

. (4 ) reporte da aiaereaoo d pretoi a(hyaliao ) drople t foraatie ai ath o opitholia l coll o @ f th o prosiaa l fcubuloo afto r U Goxpoaura . Th o rol o o f @2u=globuli n i n th o pretoi adreplo t

nephropath y wil l b o dioeuooo di ath o followin g paragraphs

. Shor t oj i cAo (9 ) reperto dtha t th o ostoa t

(au©bo r

o f tubulo o affoetod ) oad/e r

th o oevorit y (ois o @ r ohapo ) © f

£h o jpsetai adreplot o iaereaoe dafte r

a21-da y gavag o regiao a© f 5 0 t © SO

© ©g/k g TMP

5 th e reopoao e wa o P 2 OGgmoat-opGcifie , aa da© dooe-reopoao o rolatieaohi p wa s obocrvGd

. Pretoi adroplo t ovaluatio ai akidaey o o f ©al o rat o d©oe dwit h0 t o 5 0 ©g/k g TM

Pfo r 2 1 day o ohewod , however , adooe-relatG diacroas e i a t h e sea aocor o fo r droplot o (Pigur o 3 ? I * = ©ild , 2 * •= ©odorate , 3 + ° OGvar o aa d4 * <=

> ©arkodl y oovoro)

. ©vo r th o raag o ©

f dooo o otudiod , th o iaeroooo o i ath o ©oa aocor o fe r dreplot o parallolo dth o iacroaoo o i ath o porcoatag o o f

labele dcoll o ©vo r th o tota l cell o (Figur a3)

. Thuo , protoi n drople t

foraatio n an dcol l proliferatio n appoa r t o b o rolato d phenomena

. ° Mal Grat o treate dwit haaingl o doo o o f 50 0 mg/k g TM

Pohowe dahighe r ©ea aocor o fo r

protoi n droplet o tha acoatrol o (3. 4 a0. 1 v o 1. 4 & 0.1 , respectively)

. Kidne y heaogenate o obtaine dfrc © treate danimal s wer e use d t o ©eaour e th e rena l concentratio n o f @2u-globuli n usin g a aELIS A assa y wit haEOUO G

### ©onoclona l antibod y raise dtowar dpurifio dra t urinar y e2u

globulin

. Rena l coacGatratio ao f @2u-globuli n wa o ©arkGdl y iaereaoe di n t h e TMP-treato dgrou p coapare dt o tha t o f cor n oil-treate drat o (28. 1 & 2. 7 v o 9. 2 aO.S

. a g @2u-globulin/ g wo t weight)

. Moa aocoro o fo r droplet o aa drena l @2u^-globuli acoaceatratio n wGr o ao t sigaificaatl y ( p > 0.05 ) iacroaoG di afosal o rat o troato dwit h50 0 ©g/k g TMP
. laauao

- histochemistr y studio s performe duoin g th e mous e monoclona l antibod y ohowe dtha t

i awel l porfuoo dkidaoy o ebtoiao dfro © 5 0 ag/k g TMP°treate d rat o th o roaa l @2u-glebuli awo o localise dwithi ath e protoi adroploto

.
BIOCHEMICA

LEVALUATIO

NO FTH EMECHANIS

M O FTMP-INDUCE

D NEPHROPATH

Y Th e result s discusse di ath o prGviou o ooction o indicate dtha t rena l tumo r formatio n may b o relate dt o a aiacreaoo dcol l proliforatio ao f th e cell s fro maspecifi c aro ao f th o kidaoy , aa dtha t thi o rogGnorativ e proces s &ajb e rolato dt e th o protei adroplG t nephropath y ©ceuria g a t thi s sam o oit o i nth o aephroa

. @2u=globuli aappoar o t o coatribut o i n th e nephropath y oinc o th Gprotei adroplot o coatai athi o aajo r ©al o ra t

urinar y protein

. Howovor , th o aechanio ® o f it o rol o i n th e nephropath y ha o yo t t o b e detcrminod

. Th e presen t oectio n wil l sussaarig e th o reoult o o f recen t - 4 -P.74
.



### fractio n wa s associate d wit h s2u-globulin j ge l filtratio n (Sephade x G-7 5 colua a olute d wit h 6 7 ©M phosphat G buffor , p H 7.2 ) wa o use d t o separat e th o protGino
. Chromatographyo f aal o ra t kidne ycytoso l separate d ®2u

globuli n fro ©

othe r hig h an

d lo wmolecula r weigh t proteins
. Radiolabe l derive d fro ©[ HJ=TMP-troate d ©al G ra t kidne ycytoso l elute d int o tw o distinc t poako , ©n o i a th o molecula r weigh t rang e o f e2u-globulia , whic h constitute d abeu t 26 2 © f th o tota l radiolabe l i a th o eyteoeli e fractio n o r 15 2 e f th o teta l sadielaba l i n th o kidney ? materia l i a th o froctioa s o f th o firo t poa k e?@oo=roaeto d wit h th o ©onocloaa l aatibedyraioo d towar d purifie d ra t urinary®2u=globulia
. Th e ooeead poa k o f radielaba l satoria l oluto d i a th o opal l ©oloeula r weigh t raag o aa d i o though t t©repreoea t TM P aotabelitoo o Fosal o sa t kidno yeyteoeli e fraetien o proparo d fro ©animal s troato d 2 4 h r previousl ywit h [ H]°TM P rooelvo d int o oa o diotiac t pea k i a th o osal l ©olocula r weigh t rango . ThorGfero , eo-olutio a o f TMP-derive d radielabo l wit h © ^ -globuli n i o a ©al o rat-opocifi c phGaeaoaoa
. Appreaiaotof y20 2 ® f th o radielabolo d ©atGria l proooa t i a aal o ra t kidao y©yteoo l wa o aoa-dialysabl a fellewia g 1 8 h r oquiliferiu a dialysi s (100 0 d cutoff ) agaiao t 1 0 m M phoopha ^ buffer , p H 7.0
. Chromatograph yo f th o dialyso d eyteoo l @ a th e Sophado a ©°7 5 colua a ohowe d tha t th e aoa dialysabl o radielabolo d ©atGria l ee=oluto d wit h th o fraetien o eeataiaia g th o <32u°glebulia
. Equilibria ©dialyoi o e f ©al o ra t kidao ycytoso l agaias t buffo r eeataiaia g 0.1 2 oodiu a dodocy l oulfat o (SDS ) lo d t e loo o o f thi o biadiag
. SD S i o a dGtorgen t whic h deotroy o th o oeeondar yan d tertiar y otructure o © f protoia o (22) 8 ehomical o boua d t e th o protGi a vi a wea k forcG o (hydroge a boado , ioni c beads , Va a do r Uaal° o foreos ) wil l b e diooeciato d fromi t an d release d iat o th e dialyoi o buffo r upo a additio n o f SDS . T&s&o , ©u r fiadiag o iadieato d a rovoroibl o aooeciatio a botwoo a TMP
derive d radiolabe l an d s2u=globulin o Ethy l aeotat o attractio n o f th o poole d @2u-globulia-coataiaia g fraetien o o f ©al o ra t kidne ycytoso l fro m[ H]=TMP-treato d rat o recovere d ©or o tha n ©1 2 © f th o tota l radiolabel
. Ga s chroaatography-eas s spectrometr y© f th o acidifie d ethy l acetat e oatrac t ohowe d tha t 2,4,4

- trimothyl-2-pentaao l wo o th e metabolit e associate d wit h th e 82u°globulia
. Go o chromatographi c aaalyoi o o f acidifie d ethy l acetat e eatrac t prepare d fro©kidaey o o f aal o rat o expooe d by iahalatio a t o 5 0 pp a TM P o r 30 0 pp a U G ( 6 hr/day , 5 dayo/woek , 3 weoko ) alo o ideatifie d 2,4,4-tri©othyl-2

- pentano l a o th o eho@iea l boun d t o @2u-globulin § tw o otho r mino r component s (ye t t o b e identified ; thei r retentio n time s sugges t othe r branche d alcohols ) wer o alo o extracte d fro ©th e sample s prepare d fro m UG

expooo d rato . Gol.filtratio n o f kidne ycytooo l prepare d fro m50 0 ag/k g [ H]=TMP=treSto d rat o and determinatio n o f th o radiolabele d materia l i n "the @2u-globulia-eeatainin g fraction s showe d tha t 8 , 24 , 4 8 and 7 2 h r afte r dooing , th o coaeoatratio a o f boua d 2,4,4=trimethyl-2°pentaao l wa s 78. 9 o 7.2 , 118. 0 *4.3 , 101. 3 a 8. 5 an d 85. 5 *13. 8 nmol/© l o f kidne y cytoool , roopoetivoly
. Thi o oboorvatio n indicate s tha t th e 2,4,4 = trimethyl=2°pontanol=@2u-globuli n cosplG a remain s i n th e kidne yo f mal e rat s fo r a long perio d o f tim e suggestin g a fairl ystabl e associatio n i n vivo
. Cel l proliferatio n ha s boo n aoooooe d biochemically , 4 day o afte r a singl o ora l doo o (4. 4 saol/kg s equivalen t t o 50 0 mg/k g TMP) , fo r eac h o f th e known TM P metabolite©
. N o mal e rat-opecifi c increas e wa o oboorve d i n theo e conditions
. Cel l proliferatio n ha o als o bee n measured
. 1 dayafte r th o lao t o f 7 dooo o o f 0.4 4 mmol/k g 2,4,4-trimethyl-2-pentano l o r 2,4,4

- trimethyl-l-pontaaoi c acid , th e majo r urinarymetaboliteo s a oignifican t increao G abov e contro l valu e wa o observe d fo r th e 2,4, 4 = trimethyl-2

- pentanol-treate d group , bu t no t fo r th e 2,4,4-trimethyl-l-pentanoi c acid

treate d group . Therefore , th e metabolit e o f TM P revoroibl yboun d t o 62u

- -6

-

### exposur e t o U G vi acontaminate d drinkin g water , Colema n ej taJL . (25 ) reporte d tha t97 Z b y weigh to f th e component s i n th e water-solubl e fractio n o f

unleade d gasolin e wer e aromati ccompounds . Result s presente d i n thi s pape rshowe d tha tth e aromatic s i n U G do no tappea rt o b e implicate d i n th e nephrotoxicity , an d tha trena l tumo rformatio n ma y onl y b e amal e rat-specifi cresponse . Therefore , rena l tumo rformatio n i n humans followin g consumptio n o f drinkin g wate rcontaminate d wit h U G appear s unlikel y t o occur .
ACKNOWLEDGEMEN T 

### V e gratefull y acknowledg e Dr . Jame s S . Bus fo rhi s contributio n i n th e creatio n o f

thi s researc h program , an d Ms . Viek i L . Burnett e an d Mr . Jose f Strasser , Jr . fo rthei rexcellen ttechnica l work .

- 8 -

### 13

. D.J
. Loury , T
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. Toxicol

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. 14

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. Ligh t hydrocarbo n nephropathy

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. P.H
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. Leek , odo. , Joh aHile y an dSons,^Chiehootor , Gr

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. 15

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• 10

-

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### Breathin g Zon e Level s Of Gasolin e Vapor s Durin g Automobil e Fuelin g

Ronal d L . William

s Genera l Motor s ResearchLaboratorie s Environmenta l Scienc e Departmen t Warren , M I 48090-905 5 P.8 0


### 87-31. 1P.8 1



### 87-31. 1

INTRODUCTIO

N Human s ar e exposure d t o gasolin e vapor s i n refineries , a t bul k terminals , an d i n servic e stations
. Healt h concern s abou t gasolin e hav e historicall y focuse d o n certai n constituent s suc h a s polycycli c aromati c hydrocarbons , benzene , an

d lead additives
. Potentia l huma n healt h effect s fro mthes e component s hav e bee n extrapolate d fro manima l exposur e studie s an d hav e bee n evaluate d i n occupationa l groups . Arecen t stud ysponsore d b

yth e America n Petroleu

mInstitut e (API ) showe d tha t exposur e t o vaporise d whol e gasolin e had induce d kidneytumor s i n mal e rats, ^ although th e specifi c caus e wa s unknown
. Thi s stud

yprompte d th e automobil e industr yan d EPAt o as k th e Healt h Effect s Institut e (HEI ) t o revie wth e scientifi c literatur e relatin g gasolin e vapo r exposur e t o human , cancer . Th e HE I report, 3 

### issue d i n Septembe r o f 198 5 conclude d tha t although wholl yvaporise d unleade d gasolin e i s an anima l carcinogen , th e vapo r compositio n i n th e anima l studie s wa s sufficientl y differen t fro

mtha t t o whic h human s ar e expose d tha t "us e o f th e AP I dat a t o develo p dose-respons e relationship s suitabl e fo r huma n ris k assessmen t i s questionable
. I n addition , adequat e dat a ar e no t currentl yavailabl e t o characteris e accuratel yth e natur e an d exten t o f huma n exposure s t o gasolin e vapors. a 

### Thi s pape r summarise s an earlie r Genera l Motor s stud ytha t wa s undertake n t o measur e th e concentration s o f hydrocarbo n (HC ) vapor s i n th e breathing zon e o f a perso n fueling a vehicle. 1 

### * I n addition , a revie wi s presente d o f othe r publications.tha t addres s gasolin e vapo r exposure s during fueling
. Difference s among al l thes e publication s i n th e sampling an d analysi s technique s an

d i n th e unit s use d t o repor t exposure s ar e reconciled
. Finally , a bes t valu e fo r th e rang e o f exposure s i s derive d fromth e tota l dat a set . EXPERIMENTAL I n 1985 , fiftee n vapo r sample s wer e collecte d durin g Februaryan d 1 8 sample s durin g Augus t an d Septembe r nea r a gasolin e pum p a t th e Genera l Motor s Technica l Cente r i n Warren , Michiga n use d fo r vehicl e fueling
. Asmal l Tedla r ba g wa s fille d wit h ai r take n fromth e breathing zon e o f a perso n standing adjacen t t o th e car' s fille r pip e during onl yth e time tha t th e gasolin e pum p wa s running
. Wind speed , win d direction , an d ambien t temperatur e wer e note d befor e collectin g eac h sample
. Th e concentration s o f individua l H C wer e determine d b

yga s chromatograph y(GC ) an

d th e tota l hydrocarbo n (THC ) wa s measure d b

ysumming th e area s o f al l th e G C peaks
. Tw o backgroun d ai r sample s wer e collecte d durin g th e samplin g progra mt o verif ytha t backgroun d level s o f H C di d no t contribut e measurabl yt o th e level s o f th e gasoline-vapo r compounds
. Th e gasoline s use d fo r fueling wer e commercia l unleade d gasolines
. On e sampl e eac h o f th e winte r an d summe r fuel s wa s collecte d fromth e gasolin e pum p fo r G C analysi s an d Rei d vapo r pressur e measurements . Th e fuele d car s wer e al l 198 5 Genera l Motor s model s froma corporat e fleet
. Th e tan k fue l an

d th e dispense d fue l wer e generall yth e same , excep t fo r change s i n th e compositio n o f th e tank fue l du e t o weathering
. Th e us e o f a singl e fue l i s a n importan t consideratio n becaus e i t permit s an unconfounde d compariso n betwee n th e compositio n o f th e refueling vapor s an d th e compositio n o f th e dispense d gasoline
. Fure yan d Nage l recentl yshowe d tha t fueling vapor s ar e compose d o f abou t two-third s dispensed-fue l vapo r an d abou t one-thir d tank

fue l vapor , whic h woul d complicat e th e compariso n i f th e tw o fuel s wer e different
. Th e fuel-tank fille r pip e locatio n wa s documente d fo r eac h vehicl e becaus e thi s paramete r affecte d th e orientatio n o f th e sampling poin t relativ e t o compas s wind direction , a s indicate d in.Figur e 1
.P.8 2


### 87-31. 1

Procedur e fo r Collecting Vapo r Sample s Th e objectiv e wa s t o collec t an ai r sampl e representativ e o f wha t a vehicl e fuele r woul d breath , i n a containe r tha t coul d b e take n t o th e laborator

yfo r G C analysis
. Tedla r sampling bag s (3-lite r size , SKC , Inc. ) wer e partiall yinflate d b yai r draw

n b ya Gillia n persona l air-sampling pum p (Mode l HFS-113 , Wayne , NJ)
. Th e sampling lin e wa s clippe d t o th e lape l o f th e fueler' s coat , essentiall yi n hi s breathing sons . Th e fuele r stoo d adjacen t t o th e fille r pip e int o whic h the fuel-pum p nozzl e wa s inserted , a positio n commo n fo r al l wh o fue l vehicles
. Th e distanc e betwee n th e saapl e lin e inle t an d th e fille r pip e varie d fro mtwo t o four feet , dependin g upo n exactl ywher e an d ho werec t th e fuele r stood
. Th e sampling pum p wa s turne d o n an

d of f concurrentl ywit h th e gasolin e pump . Th e ba g thu s containe d vapor s wit h a compositio n average d ove r th e filling period
. Sinc e th e gasolin e pum p wa s equippe d wit h an automati c shut-of f valve , th e fueling rat e (abou t 8 gal/min ) wa s th e same fo r al l th e sampling periods , an d th e lengt h o f th e sampling perio d wa s proportiona l t o th e fue l volume adde d t o th e tank
. Th e sampling perio d range d fro

m3 0 second s t o tw o minutes , corresponding t o fil l volume s o f 4 t o 1 6 gallons
. Th e pump-and-ba g uni t wa s returne d t o th e laborator yfo r G C analysi s o n th e same day
. Analysi s o f Vapo r Sample s Th e vapo r sample s wer e analyze d fo r individua l hydrocarbon s wit h a Perki n Elme r Mode l 392 0 ga s chromatograph equippe d wit h a flam e ionizatio n detecto r (FID) , a singl e capillar ycolumn , a sampl e loop an d 8-por t Carl e sampling valv e fo r ga s samples , an d a syring e injectio n por t fo r liqui d samples
. Th e operating condition s ar e give n i n Tabl e I . Vapo r sample s an

d calibrating gase s wer e pumpe d o r flowe d int o th e 2 5 c@ ^ sampl e loop an

d injecte d ont o th e colum n wit h th e Carl e valve
. Th e detecto r outpu t wa s connecte d t o a Hewlett-Packar d Mode l 3390 Aintegrator , whic h printe d a chromatogra man d a tabl e o f peak area s an d retentio n times
. Th e chromatograp h wa s calibrate d twic e dail ywit h tw o cylinder-ga s mixture s (Scot t Specialit yGases) , on e containing 5 ppm V o f benzen e an

d th e othe r containing abou t 1 5 ppm

V eac h o f propane , n-butane , n-pentane , n-hexane , an d n-heptane
. Th e cylinde r gase s wer e precision-dilute d abou t 10

- an d 20

- fol d wit h HC-fre e air
. Th e calibratio n facto r wa s take n a s th e averag e o f th e ratio s o f th e are a count s divide d b yth e dilute d concentratio n o f eac h hydrocarbo n an d varie d betwee n 2190 0 an d 2390 0 are a count s pe r ppa C fro mda yt o day
. Th e individua l hydrocarbo n concentration s wer e calculate d fromth e dail y calibratio n factor , an d th e individua l pea k area s an d wer e summe d t o obtai n th e tota l hydrocarbo n concentration
. Th e individua l hydrocarbo n peak s wer e identifie d b ythei r retentio n times , b yanalyzing man

ycylinder-ga s mixture s containing know n hydrocarbon s an

d b y preparing othe r mixture s fro mpur e hydrocarbon s an d hydrocarbon-fre e air
. No t al l th e hydrocarbon s i n th e fueling sample s hav e bee n identified , bu t al l th e mai n peak s hav e bee n classified
. Tha t is , th e principa l hydrocarbo n o r hydrocarbon s i n eac h pea k hav e bee n identified , although some mino r one s ma y als o b e present . Th e identificatio n i s reasonabl ycomplet e an d definit e through th e C g compounds ; th e Cg olefin s ar e no t individuall yidentifie d sinc e th e retentio n time s o f al l 1 7 isomer s hav e no t bee n determined
. Onl yth e majo r C*

? an d C g paraffin s hav e bee n identified ; th e C yan d C g olefin s ar e relativel ymino r constituents
. Fou r naphthene s (cyclopentane , methylcyclopentane , cyclohexane , an d methylcyclohexane ) an d si x aromatic s (benzene , toluene , ethylbenzene , an d th e xylenes ) complet e th e lis t o f identifie d hydrocarbons
. Sinc e n o peak s wer e see n beyond o-xylen e a tP.8 3



### 87-31. 1

5 1 minute s retentio n time , w e believ e tha t essentiall yal l th e hydrocarbon s i n th e fueling vapor s wer e detected
. Analysi s o f Dispense d Fuel s Asampl e o f th e winter-grad e gasolin e wa s take n fromth e gasolin e pum p o n February1 5 an

d wa s analyze d fo r individua l hydrocarbon s using th e Perki n Elme r chromatograph
. Asampl e o f th e summer-grad e gasolin e wa s take n fro mth e pum p o n Augus t 2 2 an

d wa s analyze d using a differen t chromatograph , whic h provide d bette r separation s o f th e highe r molecula r weigh t hydrocarbons
. I n bot h cases , th e fue l analyse s extende d through Q*n anc 

### ^ include d essentiall y al l th e constituent s i n th e gasolines
. Th e gasolin e composition s wer e deduce d fro mth e chromatogram s an d th e integrato r count s fo r eac h peak
. Th e percentag e o f eac h constituen t o n a carbo n basi s wa s calculate d a s the rati o o f th e integrato r count s fo r tha t pea k t o th e summatio n o f count s fo r al l th e integrate d peaks
. Becaus e o f th e us e o f differen t chromatographs , som e H C pair s (notabl yp-xylen e an d m-xylene ) wer e separate d i n th e summe r gasolin e tha t wer e no t separate d i n th e winte r gasolin e o r i n an yo f th e vapo r samples
. Thus , whe n th e summe r gasolin e an d refueling vapo r composition s ar e compared , some grouping s o f th e gasolin e dat a wil l b e necessary . Th e Rei d vapo r pressur e o f th e winter-grad e gasolin e wa s 13. 7 psi , whil e tha t o f th e summer-grad e gasolin e wa s 10. 4 psi
. RESULT S AND DISCUSSION Bot h fue l volatilit yan d weathe r condition s wer e suspecte d a s factor s tha t migh t influenc e th e exposur e o f a vehicl e fuele r t o H C vapors . I n mos t U.S . locations , fue l compositio n i s adjuste d seasonall yt o matc h volatilit ywit h th e expecte d ambien t temperature . Consequently , w e designe d ou r samplin g progra mwit h bot h winte r an d summe r segment s t o includ e th e wides t rang e o f possibl e fue l an

d weathe r effects
. A t th e outset , w e wer e no t certai n whic h factor s woul d b e mos t importan t t o monitor , an d therefore , mad e som e procedura l change s during th e program . Sinc e th e sampling time wa s proportiona l t o th e amoun t o f fue l adde d t o th e car , fill s o f les s than five gallon s di d no t provid e enough sampl e t o adequatel yflus h th e G C loo p befor e injection
. I n thos e cases , on e o r two additiona l car s wer e sample d t o increas e th e sampl e volume
. Thi s modificatio n shoul d hav e n o effec t o n th e results , sinc e th e fue l an d weathe r wer e identical
. Also , th e individual s collecting th e sample s wer e differen t i n th e tw o segments , whic h caused othe r difference s tha t wil l b e discussed
. A s a result , th e tw o dat a set s wil l be treate d i n paralle l fashio n an d intercomparison s wil l b e mad e onl ywhe n they ca n be don e witj i confidence
. On e o f th e importan t factor s affecting th e TH C concentratio n o f the sample s wa s th e exac t positio n o f th e fueler ; hi s bod yorientatio n an

d ho w clos e th e samplin g prob e wa s t o th e fille r pipe
. Thi s wa s reflecte d i n th e las t fou r sample s o f th e summe r study , tha t wer e collecte d whil e the fuele r leane d ove r an

d graspe d th e fue l pum p nozzl e wit h hi s righ t hand , jus t a s though th e pum

p nozzl e di d no t hav e a trigger-latching mechanism
. Thi s positio n require d th e fuele r t o turn hi s bod yperpendicula r t o th e car , creating a huma n shiel d t o a westerl ywind
. Th e averag e TH C fo r thes e four sample s wa s 89 0 ppmC , wherea s the averag e TH C fo r th e firs t eigh t sample s o f th e summe r study , wit h simila r win d speed s an

d directions , wa s 7 1 ppmC . Al l th e sample s i n th e winte r stud ywer e collecte d i n th e shielde d position , whil e th e distance-to-filler-pip e variatio n wa s withi n the ergonomi c rang e i n th e genera l population
. Fo r safet ypurposes , Michiga n an d some othe r state s requir e tha t th e trigger-latching mechanis mo n th e pum p nozzl e be remove d a tP.8 4



# — n 87-31. 1

self-serv e stations , thu s forcing th e motoris t t o kee p hi s han d o n th e nozzl e t o keep fue l flowing
. Th e effec t o f wind spee d an d directio n o n TH C i s examine d i n Figur e 2
. Mos t o f th e sample s wer e collecte d wit h th e wind directio n fro mth e quadran t centere d o n th e west , bu t thos e sample s spanne d th e entir e concentratio n range . Similarly , wind speed , whic h range d fromles s tha n on e t o four meter s pe r second , di d no t systematicall yaffec t th e TH C values
. I a fact , some o f th e highes t TH C sample s wer e collecte d whe n th e refuele r wa s standing upwin d o f th e fille r pip e wit h a measure d wind velocit yo f 1. 5 s/s
. Apparently , edd y current s wer e create d b yth e sampling person' s bod ysn d th e vehicle , whic h wer e sufficien t t o caus e thes e high concentration s a t th e sample-lin e inlet
. Simila r effect s hav e bee n observe d i n laborator ysituation s i n whic h fume hood s fai l t o protec t worker s fro mpoin t source s i n fron t o f them. 0 

### " 8 

### Fo r subtl e difference s i n bod yorientatio n t o hav e suc h larg e effect s o n th e TH C exposures , th e concentratio n gradien t i n th e vicinit yo f th e fille r pip e mus t b e ver ygreat . Stron g gradient s ar e als o indicate d b yth e compariso n o f th e TH C concentration s o f th e refueling vapors , a fe whundre d part s pe r million , wit h thos e i n th e fuel-tank head space , abou t 5 0 percen t b yvolume, ®o r almos t 100 0 time s th e value s observe d i n th e breathing zon e samples
. Compositio n o f Fuelin g Vapor s Th e hydrocarbo n compositio n o f th e fueling vapor s di d no t chang e greatl y fro msampl e t o sampl e withi n eac h seasona l stud yan d wa s dominate d b

yth e ligh t paraffins
. Tabl e I I list s th e percen t b ymas s (carbon ) o f 1 2 o f th e mos t abundan t hydrocarbon s i n th e fueling vapors . Th e greates t differenc e betwee n th e winte r an d summe r sample s wa s th e highe r n-butan e conten t i n winter , whic h i s consisten t wit h th e highe r vapo r pressur e o f th e winter-grad e gasoline
. N-butane , isopentane , isobutane , n-pentane , an d propan e ranke d 1 t o 5 i n abundance , an

d combine d constitute d abou t 8 2 percen t o f th e tota l i n winte r an

d abou t 7 3 percen t o f th e tota l i n summer . Th e othe r seve n hydrocarbon s show

n i n Tabl e I I wer e al l presen t i n large r amount s i n th e summe r sample s tha n i n th e winte r samples
. Th e representativ e ligh t olefins , trans-2-buten e an

d 2-methyl-2-butene , wer e eac h presen t i n amount s o f 1 t o 2 percent . Th e dominan t Cg , C^ , an d C g paraffin s wer e 2-methylpentane , 2

- methylhexahe , an

d 2,2,4-trimetnylpentane
. Th e dominan t aromatic s wer e benzen e an d toluene ; theywer e presen t a t abou t 0. 5 percen t i n th e winte r saaple s an d slightl yove r 1 percen t i n th e summe r samples
. Together , thes e 1 2 hydrocarbon s constitute d abou t 8 4 percen t o f th e fueling vapor s b ymas s (carbon ) i n summe r an d 8 8 percen t i n winter
. Propan e was, , th e mos t variabl e hydrocarbo n presen t i n amount s greate r tha n I . percent , a s judge d b yit s coefficien t o f variatio n (Tabl e II)
. Propan e i s los t fro

mgasolin e mor e readil ytha n ar e othe r hydrocarbon s becaus e o f it s extreme volatility
. Thi s phenomeno n i s calle d weatherin g an d affect s th e gasolin e i n bot h automobil e fue l tank s and , t o a lesse r extent , i n undergroun d storag e tanks
. Th e othe r ligh t paraffin s an d ligh t olefin s ar e als o affecte d b yweathering , bu t t o a lesse r degre e tha n propane . Relationshi p Betwee n Fuelin g Vapo r an d Gasolin e Compositio n I t woul d b e expensiv e t o determin e th e exposur e o f fueler s t o vapor s fro m gasoline s o f man

ydifferen t compositions
. Therefore , i t i s importan t t o establis h relationship s betwee n fueling vapo r compositio n an d gasolin e composition
. Tabl e II I provide s a compound-by-compound compariso n i n mas s (carbon ) percen t unit s o f th e fuel s an d vapor s fro

mth e presen t study
. Th e resultan t vapor-to-liqui d rati o (V/L ) wil l b e greate r tha n 1. 0 fo r th e mor eP.8 5 

### P l 87-31. 1

volatil e hydrocarbons , whic h constitut e a large r percentag e o f th e TH C i n th e vapo r tha n theyconstitut e i n th e liqui d gasoline
. Thus , V/ L expresse s the vapo r enrichmen t facto r fo r eac h compound
. I n Figur e 3 , th e V/ L enrichmen t factor s ar e plotte d agains t th e norma l boiling poin t fo r eac h hydrocarbon
. I n th e winte r study , th e fueling vapo r wa s enriche d fo r al l component s wit h boiling point s belo w100°F
. I n th e summe r study , th e fueling vapo r wa s enriche d fo r al l component s wit h boiling point s belo wabou t 125°F
. Thi s differenc e i s apparentl ydu e t o th e lowe r ambien t temperatur e i n winter , sinc e th e temperatur e o f th e dispense d fue l (stored underground ) i s relativel y constan t year-round
. Th e magnitud e o f th e enrichmen t factor s - fro mles s than 0.0 1 t o ove r 1 0 - illustrate s th e dramati c differenc e i n compositio n betwee n th e fueling vapor s an d th e liqui d gasoline
. Fue l component s heavie r tha n th e xylene s wer e no t foun d i n measurabl e concentration s i n th e fueling vapors . Compariso n wit h Othe r Studie s Severa l studie s o f gasolin e vapo r exposur e hav e no wbee n published
. Although eac h o f th e studie s ha s a uniqu e combinatio n o f sampling procedure , analysi s method , reporting method , an d tes t variables , carefu l examinatio n an d interpretatio n wil l sho wtha t th e result s d o no t diffe r greatly
. I n ou r ow n study , w e observe d tha t th e concentration s o f tota l H C range d fro m4 t o 321 0 ppmC , eve n though th e onl yidentifiabl e variable s wer e th e win d spee d an

d directio n an

d th e bod

yorientatio n o f th e fueler
. Th e aea n valu e fro mth e combine d winte r an

d summe r program s wa s 11 0 ppmC , wherea s th e 90t h percentil e valu e wa s 198 0 ppm

C becaus e a fe wsample s had vary high concentrations
. I f a singl e valu e mus t b e selecte d t o represen t a distributio n o f thi s kind , Haider , e t al
. ® suggeste d using th e geometri c mean , whic h w e wil l adopt
. I n ou r study , w e electe d t o us e ppm C a s ou r reporting unit s becaus e th e flame ionizatio n detecto r effectivel ymeasure s th e carbo n conten t o f th e ga s strea m being analyzed
. Othe r units , suc h a s volume concentration , ca n b e compute d fo r individua l compound s o r fo r know n mixture s o f HC . However , mas s concentratio n unit s o f mg/m ^ mee t th e sam e criteria , and , i n addition , ar e commo n exposur e unit s fo r th e worke r environment
. Usag e o f th e ter mppm V i s mos t usefu l fo r expressing th e vapo r phas e concentratio n o f individua l HC , bu t i t i s an ill-define d ter mfo r gasolin e vapo r mixture s whos e compositio n i s no t 

### fixed
. Th e report s o f si x independen t studies*'10-1 4 yjj j b e criticall yreviewed
. Onl ysample s collecte d a t th e breathing zon e o f person s filling vehicle s wil l b e considered
. Th e collectio n an d analysi s method s wil l b e adjuste d t o a commo n basi s whereve r tha t i s possible
. Sinc e th e monitorin g time varie d fro m 2 mi n t o 8 h , al l th e result s wil l be adjuste d t o actua l filling time , t o eliminat e minu€e s an d hour s fo r whic h th e perso n monitore d wa s distan t fro m th e fille r pipe
. Al l th e reporte d value s wil l b e converte d t o mg/m * units , bu t th e individua l value s wil l b e preserve d t o permi t th e calculatio n o f geometri c means . Th e reporte d value s ar e displaye d i n Tabl e TV , along wit h brie f descriptor s o f th e parameter s tha t wil l b e adjusted
. Kearne yan d Dunham " provide d th e bes t dat a fo r determining th e actua l filling time during an 8-hour wor k shift
. Pum p islan d value s woul d depen d o n th e siz e o f th e servic e station , s o theyca n b e subtracte d a s par t o f th e background
. Sinc e full-servic e attendant s spen d mos t o f thei r time distan t fro mth e fille r pipe , theyar e expose d t o filling vapor s fo r onl yabou t 2 6 mi n i n an 8-hou r shift
. Haider , e t al
. " foun d tha t C ^ an

d C c H C accoun t fo r abou t 75 % o f the tota l fueling vapor , s o thei r Cg + 

### result s mus t b e multiplie d b y4 t o estimat e th e tota l exposures . Thei r direc t compariso n o f diffusio n badge s wit h charcoa l

### 87-31. 1

tube s indicate

dtha t th

e diffusio

nbadge s gav

e incorrec t values , abou t twice thos e of th

e charcoa l tubes . Clayto nEnvironmental's* * 13-statio nstud y showe dtha t th

e average self-servic e fil l i s 1 0 gallons , whil e 8 gal/mi ni s a goo d average value fo

r th e fuelin

g rate . Bond , et al. " designe d a samplin

g rack tha t coul db

e reproducibl y

positione

d adjacent t o a ca

r s o tha t th

e breathing-zon

e samplin

g point wa s unobstructed . Unfortunately , th e result s of Tironi , et al. 4 

### indicate dtha t shieldin

g b y th e bod y of th e samplin

g perso n significantl y

affecte

d th e breathin

g zon e concentratio

nof vapors , obscurin g th e expecte

deffect s of win

dspee d an d win d direction . CONCLUSION S Mucho f th

e disparit

y amon g th e reporte

d result s ha s bee

nremove

d b y th e adjustment s describe dabove . Th

e exposure range s fro m eac hpublicatio

nhav e bee npreserve

dthroug

hth e adjustment s (se

e Tabl e IV) . Th

e wid e range s represent th

e rea l variabilit

y induce d b y fue l composition , temperature , an d wind . Th

e geometri c mea

nvalues , however , al l fal l betwee n4 7 mg/n

r an d 14 8 mg/m^ , bothreporte db y Tironi , et al. Th e clos e agreement amon g th e mea nvalue s strongl y

suggest s tha t th

e studie s t o dat e hav e reliably quantifie

d th e breathin

g zon e level s of gasolin e vapor s durin

g automobil e fueling . Usin

g a mea nvalu e o f 10 0 mg/m ' an

d th e average compositio nvalue s fro m Tabl e II , th

e bes t estimat e fo

r th e fuelin

g exposure t o twelv e of th e major individua l HC i nppm V i s show

ninTabl e V . Thes e exposure level s occu r durin g th e fillin

g perio d only , an

d hav e a duratio

nof on e minut e per eight gallon s of gasoline pumped . Considerin g th e 0.2 0 pp

m winte r valu

e fo r benzene , a

neight-gallo

nfil l woul d hav

e t o b e repeate

d 120,00 0 time s t o produc e a benzen

e exposure equivalent t o th e occupational-exposur e limit , 4 0 hour s a t 1 0 ppm . Th e digeste

dresult s of sixindependent studie s provid e th e healtheffect s communit

y wit ha reliabl e characterizatio nof th e natur e an

d exten t of huma

n exposure s t o gasolin e fuelin

g vapors .- P8 7



### 87-31. 1

REFERENCE S 1 . "Estimatio

nof th e publi c healthris k fro m exposur e t o gasolin e vi a th

e gasoline marketin

g system, " U.S . Environmenta l Protectio nAgency , Office of Healt han

dEnvironmenta l Assessment , Offic e of Ai r Qualit y Plannin

g an dStandards , Jun

e 1984 . 2 . H . N . MacFarland , C . E . Ulrich , C . E . Holdsworth , D . N . Kitchen , W . H . Halliwell , an dS . C . Blum , " A

chroni c inhalatio

nstud y with unleade d gasolin

e vapor, " J . Am . College Tox. . 3 , 23 1 (1984) . 3 . "Gasolin

e vapor exposure an d huma ncancer : evaluatio nof existin

g scientifi c informatio nan d recommendation s fo r future research, " Health Effect s Institute , Repor t of th e Institute' s Healt hRevie w Committee , September 1985 . 4 . G . Tironi , G . J . Nebel , an d R . L . Williams , "Measurement of vapo r exposur e durin

g gasolin

e refueling, " Societ y of Automotiv

e Engineers , Pape r No . 860087 , Februar y 1986 . 5 . R . L . Fure

y an dB . E . Nagel , "Compositio no f vapor emitte

d fro m a vehicl e gasoline tan

k durin g refueling, " Societ y of Automotiv

e Engineers , Pape r No . 860086 , Februar y 1986 . 6 . K . J . Capla

nan dG . W . Knutson , "Th e effec t of roo

m ai r challenge o

nth e efficienc

y of laborator

y fum e hoods, " ASHRA

E Transactions , 83 , 14 1 (1977) . 7 . K . J . Capla

nan dG . W . Knutson , "Laborator y fum e hoods : a performanc e test, " ASHRA

E Transactions , 84 , 51 1 (1978) . 8 . K . J . Capla

nan dG . W . Knutson , "Laborator y fum e hoods : influenc e o f roo m ai r supply, " ASHRA

E Transactions , 84 , 52 2 (1978) . 9 . D . E . Seizinger , W . F . Marshall , F . W . Cox , an d M . Boyd , "Vehicl e evaporativ

e an dexhaus t benzen

e emission s a s influence d b y benzen e conten t of gasoline, " Societ y of Automotiv

e Engineers , Pape r No . 860531 , Februar y 1986 . 10 . C . A . Haider , G . S . VanGorp , N . S . Hatoum , an d T . M . Warne , "Gasolin e vapor exposures . Par t I . Characterizatio nof workplac e exposures, " Am . Ind . Hyg . Assoc . J. . 47,(3 ) 164(1986) . 11 . C . A . Kearne

y an dD . B . Dunham , "Gasolin e vapor exposure s a t a hig h volum e servic e station, " Am . Ind . Hyg . Assoc . J. , 47(8 ) 535(1986) . 12 . Clayto

nEnvironmenta l Consultants , Inc. , "Gasolin e exposure stud

y fo r th e America nPetroleu

m Institute, " CEC Jo bNo . 18629-15 , Augus t 1983 . 13 . A . E . Bond , V . L . Thompson , G . C . Ortman , F . M . Black , an d J . E . Sigsby , "Self-servic e statio nrefuelin

g exposure study, " U.S . EPA , 1985 . 14 . H . J . McDermot t an dG . A . Vos , "Servic e statio nattendant' s exposure to benzen e an d gasolin

e Yapors, " Am . Ind . Hyg . Assoc . J. , 40(4 ) 315(1979) .

### 87-31. 1

TABL E I Operatin

g condition s fo

r th e Perki n Elme r mode l 392 0 ga s chromatograph . Oal «

letecto r SPB- 1 •ethy l •illeoae coatedcapillary , 0.76 * : I.D . b y 6 0 aata nlose . Plaa e ioaisatio a
n o * rata * Celva a heliua Tota l halloa * Burne r ai r Bursa r hydroge n 6 ec/ai n S O cc/ai n SO O cc/ai n(5 0 2 4 cc/ai n(2 0 p s 

# 3
Oolua nore s teaperatur e 0 - 4 ainute e -80*C . 4-4 6 ainute e -S O t o 100' C S 4 * aiaut e 40-6 8 ainute e 100' C Injecto r teaperatur e 60* C Detecto r teaperatur e 800* C Attenuatio n* 2 Aaplifie r rane e s i 

### P* *

* Makeu pheliu a addeda t coluanoutlet .



### TABL E I I Averag e compositio no f fuelin

g vapor s —

selecte

d hydrocarbons . Mas s fCarbon ) Percen t o f Tota l H C nVdroearDone Liarh tparaffin s a-Butaa e i-Pentan e i-Butan e a-Pentan a Propan e Lia-ii tOlefin s traas-2-Butea e 3-Methyl-2-buten e Media e Paraffin s 2-Methylpentan e 2-Methylhexan e 2,2,4-Triaethylpentan e Aroatatic s Bensen e Toluen e Subtota l Winter(» ) 51.0 4 (2.6) e 14.4 1 (3.7 ) 8.0 3 (18 ) 6.1 7 (5.8 ) 8.7 4 (28 ) 1.1 6 1.4 6 1.7 4 0.3 2 0.1 6 0.6 3 0.4 7 88.3 6 flO ) (7.6 ) (7.5 ) 

# 8 8 8 8 (0.80 ) 83.0 3 (5.8 )

18.1 4 (3.6 ) 6.4 0 (10 ) 6.6 0 (5.6 ) 6.0 3 (25 ) 1.8 5 (13 ) 2.1 3 (15 ) 2.6 0 (6.5 ) 0.8 6 (6.3 ) 0.4 3 (28 ) 1.1 2 (8.0 ) 1.2 5 (16 ) 83.5 5 (1.3 )

• 1 3 saaple s - exclude f' s 6,12,1 4 (lo w THC ) • 1 4 saaple s - exclude #' s 6 (lo * THC ) an d11,1 3 (preaiu

a fuel ) e —"efficien t o f rariation(standar dderiationa s percen t o f th e aean ) 

## 9P.8 9



### 87-31. 1
TABL E II I Hydrocarbo n compositio n o f fuelin g Yaporan

d

Propaa a Isobutaa e Isobutylaa e • 1-Butaa a a-Butaa e traas-2-Buten e cis-2-Buten e 8-Methyl-l-buten e laepaataa a 1-Peataa e 2-Methyl-l-buten e a-Pentaa e trans-2-Pentea e cis-2-Penten e 2-Methyl-2-buten e 2,2-Diaethylbutan e Oydopenten e 4-Methjrl-l-peatea e Cyclopentan e 2,3-Dlaethylbutaa e C . Olefi n 2-Methylpaataa e C . Olafi a 8-Methylpantaa e C«Olefin s a-Hexaa e C - Olefin s Methrlejcloaentan e 2,2-Diaethylpentan e • 2,4-Diaathylpentan e Beasea * S , 3-Diaethylpentaa e Cyclohexaa e 2 , 3-Di»ethylpentaa e 2-Methylhexan e Cyclohexan e S-Methylhazan a C. Olefi n S-Ethylpentan a 2,2,4-Triaethy1pentan e a-Heptaa e C. Olefin s Methyleydophexaa e 2,5-Diaethylhexan e 2,4-Diaethylhexan e 2,3,4-Triaethy1pentan e Toluen e C g Paraffin s < £ Paraffin s 3-Methylheptan e 2,2,5-Triaethylhexan e a-Octaa a Bthylbaaaea e p-Xylaa e a-Xylen e o-Xyles e Vaaor * 4.1 2 7.7 6 1.3 5 81.1 9 1.2 6 1.3 0 0.1 6 14.2 6 0.7 1 1.0 5 4.9 0 1.2 7 0.6 3 1.4 8 0.1 6 0.2 7 0.6 3 1.7 7 1.0 0 0.1 7 0.8 2 0.6 1 0.6 6 0.1 0 0.3 7 0.1 8 0.2 0 0.1 3 0.0 7

0.4 0

0.0 1 0.0 2 0.0 1 

### dispense d gas o

Mas s (Carbon) fereen to f

Tota l I C Winte rStudy Casolin e 0.1 4 0.6 8 0.1 4 S.2 7 0.2 * 0.2 9 0.1 2 7.S 1 0.5 1 0.7 6 4.5 1 1.2 2 0.6 6 1.7 4 0.3 2 0.4 5 1.1 6 3.8 3 2.8 6 0.4 7 3.2 0 1.9 6 3.1 6 0.7 0 2.7 4 1.6 3 1.8 8 1.6 6 1.1 7

7.8 2

1.1 3 S.0 3 1.4 4 2S. 6

U . 2 8.7 8 6.1 9 4.6 7 4.S 6 1.2 S 1.9 5 1.S 8 1.S 8 1.0 9 1.0 4 0.9 5 0.S 5 0.6 0 0.6 0 0.6 S 0.4 8 0.S 5 0.S 7 0.2 6 0.2 6 0.2 1 0.1 5 0.1 3 0.1 0 0.1 0 0.07 8 0.05 6

0.05 2

0.00 8 0.00 7 0.00 6 6 

## Yswr b 6.S 2 9.8 3 0.9 S 80.6 5 2.0 3 2.0 7 0.S 6 18.4 5 1.0 5 1.6 6 6.4 0 1.8 9 0.9 0 2.4 4 0.6 1 0.S 1 0.2 6 0.7 4 2.5 3 0.2 1 1.4 6 0.3 3 1.0 7 1.S 4 0.6 4 0.3 1 1.0 7

0.7 7 0.3 1 0.2 8 0.2 5 0.1 6 0.1 1 1.2 3

0.0 8 0.1 6 uaae rStudy

Casolin e 0.0 6 0.7 S 0.2 7 4.0 6 0.4 5 0.4 2 0.1 7 7.S 6 O.S S 0.9 4 3.4 1 1.2 4 0.6 9 1.7 6 0.6 0 0.2 8 0.2 0 O.S S 1.0 3 0.1 2 S.S 8 0.2 1 2.2 5 0.6 2 2.1 1 2.8 8 

### i . a 0.9 0 S.1 8 0.2 1 0.1 2 0.1 6 S.4 6 0.0 6 1.6 7 0.3 6 0.2 2 1.6 7 1.0 1 . 0.6 4 0.6 6 0.S 4 0.3 3 1.0 6 12.6 3 0.4 6 1.0 2 0.8 1 0.2 6 0.5 2 1.3 6 2.7 0 1.0 3 1.5 6 V/L

C 70. 2 1S. 1 S.6 S 7.6 2 4.4 8 4.9 3 3.2 6 2.4 4

. 1.9 1 1.8 0 l.S S 1.6 2 1.8 0 1.S 9 1.2 2 1.1 1 0.7 4 0.7 2 0.7 6 1.0 0 0.6 5 0.5 3 0.6 1 0.4 7 0.4 3 0.S 4 0.S 4

0.2 1 0.2 0 0.1 6 0.2 5 0.2 4 0.1 0 0.06 5

0.05 6 0.04 3

Tota l Percen t88.6 8 67.6 8
* Winte raaapl e f

7 * Suaae raaapl e f

3 eCalculate d befor e roundin g 

### 9T77 5 70 S

1 0Kearne

y ft Dunha

m (11 )

Balder , et al . (10 )

Bond , e t al . (13 )

Tironi , e t al . (4 )

ClaytonEnviron . (12 )
McDermot t ft Vo s (14 ) 

### TABL E

Samplin g Situatio n Sel f servic e Ful l servic e attendan t Servic e plaz a attendan t

Sel f servic e simulatio n
Singl e ca r wit h saaplin g rac k Singl e ca r (winter ) Singl e ca r (summer ) 2- 7 car s i n self-servic e Attendan t filling onl y Ful l servic e 

### I V

4 4

attendan t durin g bus y perio d Ful l servic e attendan t Adjustmen t o f report e



### t io. Saaple s
1 2 8 2 1

1 1 paralle l perpendicula r 1 5 1 8 28 8 6 2

8 4 Samplin nTime

1 0 mi n 40 0 mi n

2 7 mi n 35 4 mi n 1. 5 mi n 1. 5 mi n 0.5-2. 0 mi n 0.5-2. 0 mi n 8-1 0 ni n
1 5 Bi n
1 5 mi n

48 0 mi n i

dexposure s t o

Reporte d Exposur e 3.0-63. 5 ag/a 3 1.0-14. 3 ag/m 3
1.1-130. 3 mg/m 3

24. 3 mg/m 3
6. 5 mg/m 3
84.0-15 7 ppa C
0.0-1 8 ppa C 4.0-321 0 ppa C
5.0-122 0 ppmC
2.4-69 0 ppmC g
12.1-09. 2 pp a gasolin e 1.81 , 2.3 1 pp

m gasolin e 0.42-40. 4 pp

m gasolin e 

### tankfilling Actua l Fillin g Time
1.2 5 ai n 21. 7 ai n 27. 0 ai n

3. 1 ai n 22. 0 mi n 1. 5 ai n 1. 5 ai n 0.5-2. 0 ai n 0.5-2. 0 ai n 8-1 0 ai n
1 5 ai n 1. 5 ai n

2 6 ai n •tim

e only .

Othe r Adjustaen t
Non e

Diffusio n badg e x1/ 2 Cg 4 reporte d X4 Diffusio n badg e x1/ 2 Diffusio n badg e x1/ 2 Unit s x0.5 6 Unit s x0.5 6 Unit s x0.5 6 Unit s x0.5 6 Unit s x3. 6 Unit s x2.7 5 Unit s x2.7 5

Unit s x2.7 5 BK/ B

Adjuste d Exposur e Rang e
31.2-50 8 35.0-26 5
35.0-417 0

—

— 47-8 8
0-1 0 2.2-175 0
2.8-68 0
8.7-250 0
33.2-27 3
50-6 4

21.0-205 0 3

Geoa . Mea n 10 2 8 5 12 8

10 6
5 2 5 0

14 8 4 7 14 0 11 2
5 6

no t availabl e - 1

•
!

.

0 0 ^ » 1 O J t-A t-tP.9 1



### 87-31. 1

TABL E V Bes t estimat e o f exposur e t o fueling vapors .
Componen t n-Butan e i-Pentan e i-Butan e n-Pentan e Propan e trans-2-Buten e 2-Methyl-2-buten e 2-kethylpentan e 2-Uethylhexan e 2,2,4-TrimethyIpentan e Benzen e Toluen e Winte r 

## (pp»n 21. 5 

## 4. S 3. 4 1. 8 2. 1 0.5 2 0.6 4 0.4 0 0.0 8 0.0 4 0.2 0 0.1 2 Summe r

fonmV } 13. 0 

## 6. 1 

## 4. 0 2. 2 

## 3. 3 0.8 5 0.0 3 0.7 4 0.2 1 0.0 0 0.3 5 0.3 3



## 1 2

## Attendant' s Shelte r

Cur b 87-31. 1



# N

Samplin g Location s Dependin g t\W- o n Fille r Nec k

Location s

Roadwa y

Ga s Pum p

Fig . 1 Pla n vie w o f fuelin g area .

1 3

### 87-31. 1 P.9 3

Winte rStud y N 

### •

- 4 meters/se c Summe r Stud y N 4 4 meters/se c



## + E

Fig . 2 Win

d directio n an

d velocit y durin g sampling . Number s indicat e tota l hydrocarbo n concentratio n i n ppmC .

1 4

### 87-31. 1


## 15.0 0 10.0 0 8.0 0 ( - \ o 

## 6.0 0 4.0 0
2.0 0RatioLiquidVapor 

## 1.0 0 .8 0 .6 0 .4 0
.2 0
.1 0 .0 8 .0 6 .0 4
.0 2
.0 1 .00 7 T

## r i 1 — i r



## Winte rStudy . \ #

^ aN 

## Summe r

Stud y

\

\ \ \



## I 

# ± \ 

# ,_ A 5 0 10 0 15 0 20 0 25 0 30 0

Norma l Boilin g Poin t (°F )
Fig . 3 Vapo renrichmen t factor s (V/L ) fo r

C^-C g hydrocarbon s i n fuelin g emissions

.
1 5P.9 5

§u&missio n Fe e Receive d SO TAbstract Numbe r— 

## SOCIETY© F T0XD < ABSTRAC TFOR M FO R TH E H9© S ANNUA L MEETIN G 

# HH

707 5 

# [ An y corroopondonc o regardin g you. - abatrac t muo

t rotorone o thi o number .
Roo d carefull y th o abotrac t Inotructlon o (attached) . Follo wforma t fo r heading . Origina l muo t b e o n thi s form . Loavo a doubl e spac e betwee n hoodin g an d abotract . Yost e f obotrae t rauot bo olngt o opaeod . Thor o wil l bo a charg o of 82 3 fo r an y abotrac t tha t muot b o rotyped . Loov

e n o marglno—typ o muo

t bo withi n biu o roctanglo . Slngt o opaco , oncop t fo r doubl e spac e betwee n bod y o f tes t an d outhoro . Us e typ o n o omalle r tha n 12-plteh . Proo fcorofuity . Ooodlln o tor rocolpt I n Enoeufte o Soerotory' o efflc o l o Octobo r IS , 1SQ7 . Submi t origina l an d on o copy'only , togetho r wit h you r submission chec k fo a e f82 S (n o ptfrchao o ordor o cccoptod) . POSSIBL E INHIBITION O F RENAL PHAGOLYSOSOMAL (PL ) PROTEOLYSI S B

YGASOLIN E IN

MAL E RAT : EVIDENC E FRO M IMMUNOELECTRON

MICROSCOPI C LOCALIZATION O F a - -GLOBULIN . M

J Olson 1 . , M

A Mancini 2 , BD.Garg 1 , anr j A KRoy 8 . 'Bioraed . Sci . Dept. , G M Res . Labs. , Warren , MI , a Dept . o f Biol . Sci. , OaklandU. , Rochester , MI . Sponsor : E W Le e
o

_-Globuli n(aG ) i s a protei nthough t t o b e integra l t o the developmen t o f mal e rat-specifi c nephrotoxicit y

cause db

y unleade dgasoline . A gold-labelle dsecondantibod y wa s usedt o determine the distributio no f aGwithi ncell s o f the rena l proxima l convolute dtubule s (PCT) . a G wa s localize dalmos t exclusivel y i nP L o f saline - o r gasoline-treate d(2. 0 mL/kg/d , 9 d ) male ra t kidney ; n o a Gwa s apparen t a t site s othe r tha nPL , microvill i o r endocytoti c vacuoles . Further , treatmen t o f mal e rat s wit h leupeptin , t o inhibi t cathepsi nB , cause d accumulatio no f P L identica l t o thos e observe d followin g gasolin e administratio nandrapidl y increase dthe rena l conten t o f aG . a Gi nPL o f contro l rat s wa s distribute duniformly . However , the enlarged , angula r P L characteristi c o f gasolin e andleupepti nintoxicatio ncontaine d deposit s o f anti-a Glocate dpreferentiall y ove r crystallin e P L inclusions . I naddition , numerou s PL , unreactiv e wit hanti-aG , wer e observe di n PC T o f gasoline - o r leupeptln-treate d rats . Thus , i t i s conclude dtha t a defec t o f lysosoma l proteolysi s i s induSe d b y gasolin e andresult s Insequestratio no f larg e amount s o f protein , includin g aG , i

nP L o f PCT . t . Nom a an d addroo o of firo t (prooontlng ) author: Sontef(flrot ) outhor o co n oubmt t onl y on o obotroct tot to o mooting . 

### Memo : Michae l J
. Olso n Organisation : Genera l Motor s Researc hLab s Addrooo : Biomedica l Scienc e Departmen t Warre n M I 4809 0 Cit y Staf o Zi p Cod a 

### US A Country Tolophono : < ^1 3 • 

### 986-166 7 2 . Cflemberohl p otatus : SO Tmembe r ANon-memb e Abotroct o muo t nov o o n SO Tmembe r (no t o otudon t member ) oo o n autho r o r b oponooro d b y a mombor . N aponoonrt . tft o SO Tmornbofoponoe r muo t otg n botoo :

Signatur e o f SO Tmombo r opons o 

### Eu n Wo o Le e Type d nam o o fSO Tmombo r opono o
3 . Conside r thi s abstrac t fo r selectio n fo r th e Ptonary Session :

.Ye s .N o 4 . Undergraduat e an d graduat e student s an d post-doctora l fe ' low s ar e als o requeste d f o chec k on o ofth o opace o below : Undergraduat e Graduat o Postdoctora l 5 . Th e Progra m Committe e reserve s th e righ t t o assig n paper s t eithe r a platfor m o r poofo r soooion . Ploao o Indicat o you r prefe i enc o belo wan d whethe r yo u wis h f o withdra wyou r pape r you r choic e canno t b e met :

Platfor m XPoste r

. Eithe r . Withdraw , i fchoic e canno t be me

t
S . Sofcc t fro m th o Ho

t bolo wth o oooolo n topi c tha t boo t oult o you r paper . Specify you r firo t an d secon d choic e o r specify anothe r possible sessio n topi c QTO

Q undo r "ofhor. " Specify onl y tw o choices . SESSIO N TOPIC S



### 1 1 General /

Mathod s 2 Aquatic/Environmanta l 3 . Molecular/Cellula r 4 . Biotransformation/Dispositio n 5 . Reactiv

e Intermediate s 6 . Genotox/Mutagenesi s 7 . Carcinogenesi s 8 . Reproductive/Teratolog y 9 . Cardiovascular/Rena l 1 0 i i 1 ? i a 1 4 I S i f i 1 7 if l Inhalation/Respirator y System s Hematologi c Oermal/Ocula r Hepatic/G l Syste m Neurochemistr

y Neuropatholog y Electrophysiotogy Behavio r Immun e Syste m 19 . Endocrin e Syste m 20 . Metals

/ Inorgani c Pesticide s Solvents Halogenate d Hydrocarbon s 24 . Food/Drug s 25 . Biomaterials/Medica l Devices 26 . Communication/Educatio n 27 . OTHER : 21 2 2 2 3

(On e topi c only ) Retur n thi o for m by Octobo r 16.1987 , to : Progra m Committee , % Enecutiv e Secretary , Societ y of Tomcology , 113 3 Fifteent h Street . N.W. , Suit e 1000 . Waohlngton , O.C . 2000 9 (202/293-5335) . Submi t abotrac t form , ort o copy , an d abotrac t oubmissio n fe e o f S25 pe r abstract . I f mor e tha n on e abstrac t l o submitte d I n th e sam e envelope . Indicat o numbe r enclose d o n th e lowe r righ t han d corne r o f envelope . Abstract s submitte d withou t fee s wil l b e returned .Submissio n Fe e Receive d

SO TAbstract Numbe r 

## SOCIETYO F T©XIC©LO<QV ABSTRAC TFOR M FO R TH E 198 © ANNUA L MEETtN G 

# HH

707 4 

# [An y corroopondonc o rogardln g your ] obotrae i muo

t roferonc o thi o number . J

Kaa d carofulr

y th e abotrac t Instruction s (attached) . Follo wforma t fo r heading . Origina l muot b o o n thi o form . Loov e a doubl e spac e betwee n hoodin g an d abotract . Ton t o f abotrac t muot bo oingt e opaeod . Thor o wil l b e a charg o of S2 5 fo r an y abotrac t tha t muot b e rotyped . Leav e n o margins—typ o muo

t bo withi n Wu o roctanglo . Singl e opace , oncopt fo r doubl e spac e betwee n bod y o f ten t an d authors . Us e typ e n o smalle r tha n 12-pifch . Proo f carefully . Ooodlirto to r rocoipt i n Eaocutlv o Socrotory' o of f ic o i o Octobe r 16,1907 . Submi t origina l an d on o cop y only , togethe r wit h you r submissio n chectt to o o f83 5 (n o purchao o ordor o occoptod) .

C Nomo : 

### Michae l J
. Olso n

FUE L HYDROCARBON-INDUCE D HYALIN E DROPLE T (HD ) ' • ^^t^con^SSS^SJ ^ NEPHROPATH YI N

MAL E RAT S DURIN GAGING . CV RMurty 1 , M

J Olson* , B D Garg 2 , an dA KRoy* . l Dept . o f Biol . Sci. „ OaklandU. , Rochester , MI , a Biomed . Sci . Dept. , G M Res . Labs. , Warren , MI . Sponsor ; E W

Le e Orgonlzotlon: Genera l Motor s Researc hLab s Addrooo : Biomedica l Scienc e Departmen t Youn g mal e rat s treate dwit hcertai nhydro carbons , includin g gasoline , develo pnephropath y characterize d b

y accumulatio no f HD incell s o f the rena l proxima l tubule s (PCT) . However , no informatio ni s presentl y availabl e onpossibl e age-dependen t susceptibilit y t o H D accumulation . PC T o f young ( 3 mo.) , untreated , mal e Fische r rat s ha dnumerou s H D i nthe P1 an dP 2 segments . B y 1 2 mo . o f age , the numbe r o f droplet s had decline dmarkedly ; a t 21-2 6 mo. , n o HD wer e visible . Electro nmicroscopy o f PC T o f age dmal e rat s showe dtha t phagolysosomes , equivalen t t o HD , wer e reduce di nnumber . We hav e als o show n tha t a majo r constituen t o f man y HD i s the mal e ra t urinar y

protein , ou-globulin , fo r whic hthe rat e o f hepati c synthesi s decline s durin g aging . Level s o f bot hhepati c an drena l a -globuli ni n 2 6 mo . ol dmal e rat s wer e about u1.5 $ o f th e Warre n M I 4809 0 Cit y Stat e Zi p Cod s 

### US A Country Telephone : L 

### 31 3 ) 986-166 7 2 . Membershi p status : SO Tmembe r _*l

_Non-memb e Abotroct o muo t hov o o n SO Tmombo r(no t o otudon t mombor ) oo o n outno ro rt oponooro d b yo mombor

. H oponoorod

. th o SO Tmombo roponoo rmuo t ofg n boiov» :

Signatur e o f SO Tmembe r spons o Eu nWo o Le e

young adult . Unleade d 5 d ) cause da 3-fol d conten t o f a

_-globuli n th e sam e treatmen t di d globuli ni nol drats . We majo r determinan t i n hydrocarbon-induced " nephropath y inmal e rat s an

d only-rat s whic hproduc e larg e amount s o f a 2 -globuli nar e susceptible . gasolin e (O. H mL/kg/d , Increas e i

nthe rena l i nyoung male s wherea s no t increas e rena l a. - conclud e tha t ag e i s a the developmen t o f Type d nom o of SO Tmembe r spons o
3 . Conside r thi s abstrac t fo r selectio n fo r th e Plenary Session : xVa n N o 4 . Undergraduat e an d graduat e student s an d post-doctora l le : low s ar e alo o requeste d t o chectt on e of th e space s below : Undergraduate Graduat o Postdoctora l 5 . Th e Progra m Committee reserve s th e righ t t o assig n paper s t> eithe r a platfor m o r poste r session . Pleas e Indicat e you r prefe i enc e belowan d whethe r yo u wis h t o withdra wyou r pape r i you r choic e canno t b e met :

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unde r "ofhor. " Spocify onl y tw o choices .

_ £ 1 . General/Method s 10 . 2 . Aquatic/Environmenta l 11 . 3 . Molecular/Cellula r 12 . 4 . Biotransformation/Dispositio n 13 . 5 . Reactiv

e Intermediate s 14 . 6 . Genotos/Mutagenesi s • .-•• A 15 . 7 . Carcinogenesi s 16 . 8 . Reproductive/Teratolog y 17 .

_1 9 . Cardiovascular/Rena l 1 8 SESSIO N TOPIC S Inhalation/Respirator

y System s Hematologi c Dermal/Ocula r Hepatic / G I Syste m Neurochemistr

y Neuropathology Electrophysiotogy Behavio r Immun e Syste m 2 1 19 . Endocrin e Syste m 20 . Metals

/ Inorgani c Pesticides 22 . Solvents 23 . Halogenate d Hydrocarbon s 24 . Food/Drug s Biomaterials

/ Medica l Devices Communication/Educatio n OTHER : 25 . 26 . 27 . (On o tope conly ) Rotur n thi o for m by Octobe r 16,1987 , to : Progra m Committee , % Esecutiv o Secretary , Societ y o f Toxicology , 113 3 Fifteent h Street , N.W. , Suit e 1000 . Woohlngton , O. C 20C0 S (202/293-5935) . Submi t abotrac t form , on o copy , an d abotrac t oubmloolo n fe e of S25 pe r abstract . I f mor e tha n on o abotrac t I s submitte d I n th e sam e envelope . Indlcot o numbe r enclose d o n th o lowo r righ t han d corne r of envolopo . Abstract o submitte d withou t fee o wil l b e returned .P.9 7

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## SOCOET YO F ABSTRAC TFOR M FO R TH E 18@ S ANNUA L MEETIN G 

# N ° 521 2 ®L©Ca Yr A n yc *rroopondonc o regardin g your ]

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. FUE L HYDROCARBON-INDUCE D SUBCELLULA RALTERATIONS^ - IN KIDNE YO F MAL E RATS . B D Garg 1 , M J Olso n" f L C~Li l an

dA KRoy a „ 'Bioraed . Sci . Dept. , G M .Res , Labs. , Warren , M I an d* Dept . Biol . Sci. , Oaklan dU. , Rochester , MI . Sponsor ; E W Le e Ora l administratio no f unleade dgasolin e t o mal e rat s produce s phagolysosome (hyalin e droplet ) accumulatio ninepithelia l cell s o f th e rena l proxima l convolute dtubule s (PCT ) simila r t o tha t observe d wit hDecalin , isoparaffIni c sol vent s an

dnumerou s othe r volatil e hydrocarbons . Transmissio nelectro nmicroscopy wa s use dt o study dose - an

dtime-dependen t alteration s o f rena l PC T phagolysosome s i nmale rat s treate d wit hgasolin e (0.4-2. 0 mL/kg , po , onc e daily , 1 - 9 d) . A

progressiv e increas e i nthe numbe r and siz e o f phagolysosome s incell s o f th e PC T wa s observed . Man

y o f the enlarge d(0.05- 9 ym) , angula r phagolysosome s i ntreate drat s containe d electron-dense , crystallin e Inclusion s an doccu pie d a considerabl e are a o f th e cytoplas m i n contras t t o the relativel y smal l (0.05-2. 5 wm ) roun dphagolysosome s o f controls . Thes e morpho logica l observation s paralle l biochemica l deter minatio no f dose - an dtime-dependen t accumula tio no f o 2 -globuli nafte r gasolin e exposure . Furthermore, studie s wit hgold-tagge dantibody t o <x

_-globuli nhav e demonstrate da - -globuli n localizatio nexclusivel y inth e phagolysosomes . Thes e result s sugges t tha t a commo ndefec t i n phagolysosome-turnove r induce db y hydrocarbon s i s responsible fo r the uniqu e sensitivit y o f male rat s t o hydrocarbon-induce dnephropathy . Nom o an d addroo o o f firo t (prooonflng ) author : Sont w(firot ) outttor o co n oubral t only on o obatroet to r th o mooting . Mowto : Bhagwa nD
. Gar g OrgonboMon : Genera l Motor s Researc hLab s Addrooo : Biomedica l Scienc e Departmen t Warre n M I 4809 0 Cit y Stot o Zi p Cod a USA Countr y Tolophono : < 31 3 • 986-165 2 2

. t&emberohl p otatus : SO Tmombo r_JL

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Signatur e o f SO Tmombo roponoo r Eu nWo o Le e

5

. Type d nom o o f SO Tmombo roponoo r
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. Pleao o Indicat o you rprofor
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8

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.

T .1

. General /

Method s 2

. Aquatic/Environmenta l 3

. Molecular/Cellula

r 4

. Biotransformation/Dispositio n 5

. Reactiv e Intermediate s 6

. Genoton/Mutagenesi s 7

. Carcinogenesi s 8

. Reproductive/Teratolog y 9

. Cardiovascular

/ Rena l 10

. 11

. 12

. 13

. 14

. 15

. 16

. 17

. 18

. SSSSSO N TOPIC S Inhalation/Respirator ySystem s Hematologi c Dermal / Ocula r Hepatic/G I Syste m Neurochemistry Neuropatholog y Electrophysiotog y Behavior Immun e Syste m 19

. Endocrin e Syste m 20

. Metals /

Inorgani c 21

. Pesticide s V> Rolv«nt s 23

. Halogenate d Hydrocarbon s 24

. Food/Drug s 25

. Biomaterials /

Medica l Device s 2 6 Communication/Educatio n 27

. OTHFR
- (On o topi conly ) Rotttr o thi o torm b yOctobe r13,1987 , to : Progra m Committee , % Eaocutlv o Secretary , Societ yo f Tonlcology , 113 3 Fifteent h Street , N MH.

. Suit e 1000 , Washington , D.C

. 2000 5 (202/233-5633)

. Submi t abotroc t form , on o copy , an d obotrac t oubmloolo n fe o o f 82 5 po rabotract

. I f mor e tha n on o abotrac t l o oubmltto d I n th e sam e envelope

. Indicat o niranbo ronclooo d o n th o towoir right han d corno ro f onvotopo

. Abotroct o oubmltto d withou t feo o wil l b o rotumod

.TOXICOLOG YAN D APPLIE D PHARMACOLOG Y90 . 43- 5 I (1987 )


### Accumulatio n o f a 2u 

### -Globuli n i n th e Rena l Proxima l Tubule s o f Mal e Rat s

Expose d t o Unleade d Gasolin e

MICHAE L

### J
. OLSON. * BHAGWA N

### D
. GARG, * 

### C

. V
. RAMAN A

### MuRTY. t AN D ARU N

### K
. RoY t

• Biomedical

Science Department. General Motors Research Laboratories. Warren. Michigan 48090. and tDepartment

of Biological

Sciences. Oakland

University. Rochester. Michigan 48063

Received December9. 1986: accepted February 18. 1987
Accumulatio n o f

a. u -Globuli n i n th e Rena l Proximal Tubule s o f Mal e Rat

s Expose d t o Un leade d Gasoline . OLSON . M . J. . GARG . B . D. . MURTY . C . V . R. . AN D ROY , A . K . (1987) . Toxicol. Appl. Pharmacol. 90 , 43-31 . Saturate d branched-chai n aliphati chydrocarbons . Toun d i n mo to rfuels , induc e nephrotoxicity i n mal e rats . Treatmen to f mal e rat s with unleade d gasolin e (0.04-2. 0 ml/k g bod y wt . po > fo r9 day s increase d markedl y th e numbe ran d siz e o f hyalin e (protei n resorption ) droplet s i n epithelia l cell s ofth e renal proxima l convolute d tubule s (PCT ) an d enhance d cellula rexfoliatio n athig h dos e levels . N o othe rtreatment-relate d pathologica l effects were observe d i n th e glomeruli , dista l tubules , o rmedulla . Th e rena l conten to f

a Zu globulin , amajo rurinary protei n o f mal e rats , was increase d maximall y b y abou t4.4-fol d after gasolin e administratio n (1. 0 ml/kg , po . 9 days) : n o furtherincreas e wa s observe d a thighe rdoses . . Immunoperoxidas e stainin g o f kidne y tissu e section s fo ra 2u -globuli n reveale d large accumula tion s o f antige n localize d i n man y ofth e PC T epithelia l cell s whic h containe d hyalin e droplets . Th e hepati cconten to f

a 2ll -globuli n an d its mRN A were no taltere d b y gasolin e administration . Thes e datashow , forth e firs ttime , tha ta !u -globuli n i s accumulate d i n th e kidney s o f gasoline intoxicate d mal e rat

s an d sequestere d specificall y i n som e ofth e hyalin e droplet s characteristi c o f gasoline-induce d nephropathy . A hydrocarbon-induce d defec ti n th e rena l lysosoma l degra datio n o f low-molecular-weigh turinar y proteins , rathertha n increase d synthesi s o f thes e pro teins , appear s t o caus e hyalin e drople taccumulation , cmiAcademi cPres. inc . 

### Volatil e petroleu mhydrocarbo nmixture s are produce dan dconsume dinhug e volume s a s moto r fuels , solvents , pain t

an dvarnis had

ditives , degreasin g agents , an dindustrial in

termediates

. Toxicologica

l assessment

ha s re vealeddose-dependen t

alteration s inth e morpholog y an dfunctio nof th e renal corte x o f mal e rat s following acut e an dchronic ex posure t o many of

thes e product s (Alden et al.. 1984 ; Haider

et at.. 1984 ; Phillip s an d Cockrell

. 1984 ; Phillip s an dEgan
. 1984 ; Garg etal.. 1986 ; Via

u etal.. 1986)

. Recently , th e importanc e o f

understandin g hydrocar

bon-mediate dnephropath y wa s increase d greatly b y th e observatio ntha t

rat s expose d fo r 1 8 month s o r

longe r t o wholly volatilize d unleade dgasolin e exhibite da nincrease din

cidenc e of

malignant

renal neoplasi a(Mac

4 3 

### Farlan det al.. 1984)

. Th e occurrenc e of tu
mor s was sex - an dspecies-specific ; mal e rat s only exhibite dtreatment-relate drena l neo plasi a(MacFarlan det

al.. 1984)

. Analysi s of whol e gasoline an d2.2.4-trimethylpentane

. a potentl y nephrotoxi c componen t o f gasoline , inshort-termassay s fo

r mutagenesi s o r

DN A damag e ha s produce dequivocal o r negativ e result s (Farro w et

al.. 1983 : Lour

y et al.. 1986 : Richardso net

al.. 1986)

. Thus , th e mechanism s b y whic hthes e agent s induc e over t nephrotoxicity are bein g studie dinth e hop e that a nepigeneti c toxi c event

which stimulate s tumo r

formatio nma

y b e identi

fied. Th e primar

y site of toxicit y of unleade d gasoline , je t

fuels , methy l isobuty l ketone

. Cio-C, 2 isoparaffinic solvent

. Stoddardsol

0041-008X/8 7 $3.0 0 Copyngh t© 198 ? by Academi cPresi . Inc . Al l ngh u o f

reproductio n in any for

m reserved .a 2ll -GLOBULI N IN GASOLIN E NEPHROPATH Y 

### 4 5 1968 ) o rimmunostaine d fora 2u -globuli n with th e avi din-bioti n procedur e (Hs u et at.. 1981) . Th e Mallory' s stai n solutio n consiste d o f (w/v ) 1 % phosphotungsti c acid . 2 % orang e G . 1

% wate rsolubl e anilin e blue , an d 3 % aci d fuchsi n in distille d water . Tissues , hydrate d with xylen e an d grade d alcohol s t o water , were immerse d fo r 2 t o 4 mi n i n th e stainin g solution , rinsed i n water , an d dehydrate d i n grade d alcohol s an d xylene . Localizatio n o f a 2u -globuli n i n deparaffinize d tissu e section s wa s ac complishe d afte rrehydratio n an d incubatio n i n asolu tio n o f 3 % norma l goa tserum . 3 % bovin e serum , an d 2 % gelatin . Polyclona l anti-cr2u -globuli n (rabbi tIgG ) wa s applie d fo r3 0 rain, an d th e tissue s were washe d with phosphate-buffere d salin e (PBS ; 0.1 4 M NaC l 0.0 1 M Na-phosphate . p H 7.4) . furthe rincubate d fo r3 0 mi n with biotinylate d anti-rabbi tIgG (goa tIgG) . an d washe d with PB S again . A fina

l 30-mi n incubatio n with freshly prepared avidin-bioti n reagen t(Vecto rLaboratories . Burlingame . CA ) wa s followe d b y extensiv e washin g i n PBS . Th e section s were the n place d i n 0.0 5 M Tris-HCl , p H 7.0 . containin g 0.001 4 M 3.3'-diaminobenzidin e te trahydrochlorid e an d 0.00 3 M hydroge n peroxid e fo r15 min . Th e reactio n wa s terminate d with a15-mi n rinse i n runnin g wate ran d th e section s wer e counterstaine d with Harri s hematoxylin . Dot-blot assay ofa. a -globlin mRNA in liver. Tota l he pati cRNA , extracte d with pheno l an d sodiu m dodecy l sulfate , wa s use d a s th e startin g material t o isolate . poly(Af-containin g RN A by oligo(dT><elluIos e chro matograph y (Avi v an d Leder . 1972) . Seriall y dilute d poly(A)*-containin g RN A wa s spotte d o n nitrocellulos e filters (Thomas . 1980) , dried , an d hybridize d with "P labele d a 2u -globuli n cDN A (Ro y et al.. 1982) . Bindin g o f th e prob e wa s determine d b y autoradiograph y usin g KodakX-ray film an d quantitaie d with soft-lase rscan nin g densitometr y (Biome d Instruments . Fullerton , CA ) o f th e develope d film . Radioimmunoassay of a, u -globulin in kidney and liver. Th e cytosoli cfraction s o f kidne y an d live rhomoge nate s were prepare d an d assaye d fora 2u -globuli n b y dou ble-antibod y RI

A a s describe d b y Ro y (1977) . Th e quan tit y o f a 2u -globuli n i n th e supernatan tfractio n wa s ex presse d relativ e t o tota l protei n (Lowry et al.. 1951) . 

### RESULT S Characterization of

Test Material Ga s chromatographi c analysi s o f th e gaso lin e administere dt o rat s showe dacomposi

tio nb y weigh t of abou t 51 % paraffins

. 5

% naphthenes

. 10

% olefins , an d26% aromati c compound s (Tabl e 1)

. Th e remainin g 8 % of th e material was no t

identifie db

y cochroma

tography wit

hknow n standards

. Ofth e paraf

TABL E 1

SUMMAR Y O F HYDROCARBON S I N UNLEADE D GASOLIN E USE D FO RORA L ADMINISTRATIO N Hydrocarbo n clas s /t-Paraffins Is o paraffins Naphthene s Olefin s Aromatic s Total s Carbo n chai n

length detecte d C t-C,. C-C, , Cj-C u C 4 -C , Q-C, : Isomers

identifie d 1 4 5 8 40 7 2 5 3 23 7 Weigh t % 16. 4 34. 3 5.4 10. 0 26. 4 92. 5 Note. Gasolin e sample s use d forindividua l experi ment s wer e analyze d b y ga s chromatograph y a s de scribe d unde rMaterials and Methods. Typica l results were from analyse s o f

thre e lot s o f

gasoline . 

### finic constituent s abou t

68 % wer e saturate d branche dcarbo n chai n compound s (isopar

affins) , suc ha s 2,2.4

- an d2,3.4-trimethylpen

tane , whichhav e been identifieda s poten t ne phrotoxicant s (Haider

et al.. 1984)

. Light Microscopic Histology In kidney s o f

contro l rats , hyalin e droplet s wer e observe dinth e epithelial cell s o f afe w scattere dPC T (Fig

. IA)

. Th e droplet s wer e clearly visibl e onl y in tissu e section s treate d wit hMallory' s Heidenhai n stain ; insection s staine dwit

hhematoxyli n an deosin

. th e hya

lin e material appeare da s refractile

. slightly acidophili c inclusion s i

n th e cytoplas mof PC T epithelial cells

. No t

all th e epithelial cell s of any single PC T containe dhyalin e droplets

. Likewise , no t

all PC T containe depithelial cell s wit hhyalin e material (Fig

. 1 A)

. Affecte d tubule s wer

e arrange din band s o r

ray s ori

ente dfro mth e renal capsul e t o th e oute r zon e o f th e medulla

. Th e droplet s wer e small (ca

. 0. 5 umdiameter) , unifor

minsize , an dexhib

itedaroun do r

slightl y ova l cross-sectiona

l profile

. However , occasionally asingle PC T epithelial cell was observe dengorge dwit hhy

alin e droplet s whichappeare dcoalescent

. No hyalin e material was note di n th e cytoplas m of epithelial cell s linin g the distal tubule s o r collectin g ducts

.P. 1

o 2ll -GLOBULI N IN GASOLIN E NEPHROPATH Y 4 7 

### Treatmen t

of rat s wit h0.04-2. 0 m

l gaso line/kg orally fo

r 9 day s produce damarke d increas e in th e numbe r

o f hyalin e droplet s in th e PC T epitheliu m(Fig

. IC)

. Bot hth e num ber o f tubule s wit

hhyalin e droplet s an dth e numbe r o f hyalin e droplet s per

epithelial cell wer e increase din compariso n t o controls

. Th e populatio no f

hyalin e droplet s induce d b y administratio n of

gasolin e was heteroge

neou s wit hbot hfin e (ca

. O. S Mt n diameter ) an dcoars e (1- 3 fim) droplet s visible

. Th e large r droplet s frequently possessedirregula r profiles

. Occasionally , th e continuity o f th e PC T epitheliu mwas disrupte db y foca l los s of cells , whichwer

e shedint o th e tubul e lu

men . In man y microscopi c fields , lumin ao f some distal tubule s wer

e fille dwit hgranula r o r amorphou s material whic hstaine dposi

tivel y withth e Mallory' s Heidenhai n method

. As in th e salin e controls , glomerul i appeare dnonna l wit

hrespec t t o siz e an dcel

lularit y o f th e vascula

r tuf t an dthicknes s of endothelial membranes

. Cell s linin g distal portion s of

th e nephro nwer

e morphologi

cally normal

. Histologi c analysi s of

renal tissue s sug gestedtha t

dose s fro m0.04 t o 1.

0 m l gaso line/k g cause dgrade dincrease s i n th e accu mulatio n of

hyalin e droplets

. However , sign s of over t nephrotoxicit

y sucha s epithelial cell exfoliatio n wer

e visibl e onl y at

dose s o f 0. 4 ml/kg o r more administere dfo

r 9 days

. Renal and Hepatic a 2u 

### -Globulin Content Followin g 9 day s o

f treatment , th e kidney s o f rat s administere d0.04 ml gasoline/kg o r more containe dsignificantly more a 2u 

### -globu

lin tha ncontrol s (Fig

. 2)

. Administratio n of 0.00 4 ml gasoline/kg o r

les s di dno t

alte r th e concentratio n of

a-. u 

### -globulin detecte di n th e kidney s o f treate drat s compare dt o controls

. Th e renal accumulatio n of a 2u 

### -globuli n oc curre din adose-responsiv e manne r only in th e rang e o f

dose s between0.0 4 an d1. 0 ml / k g (Fig

. 2)

. Maximal accumulatio n of a 2u 

### globuli n(440 % of

control ) occurre dfollow

in g administratio n o

f 1. 0 m l gasoline/kg

. IS O

8 i »

" 3 o 10 0

7 5

T?- M a

- a c 2 S -fr-^- 1 c S o a. o > E B> 3 c 3 n o O 3 <M a 2 S < s Q . < B X 0 4 « 10- * 4 i KT3 4 « 10-2 « , io-<

Gasolin e Dos e (mL/kg ) FIG . 2 . Accumulatio n o f a !u -globuli n i n th e kidne y a s afunctio n o f

gasolin e dose . Gasolin e o rcor n oi l vehicl e wer e administere d b y gavag e onc e dail y fo r9 consecutiv e days . Eightee n hour s afterth e final administratio n o f gas oline , kidney (• ) an d live r(O ) tissue s wer e sample d fo r RIA determinatio n o f

a 2u -globulin . Dat aare plotte d a s mea n ±S D a 2ll -globuli n content , n • » 3- 7 rats pertreat men tgroup . ••/

» < 0.0 1 compare d t o contro l (Dunnett' s test) ; ft p < 0.0 1 compare d t o nex tlowes tdos e (Dun cans's test) . 

### Higher dose s o f gasoline (i.e. , 2. 0 ml/kg ) di d no t caus e greater

renal accumulatio nof

thi s protein

. Th e liver s of

contro l an dgasoline

treate drat s di dno t

diffe r significantl

y ina 2u 

### globuli n conten t

(Fig

. 2)

. Further , dot-blo t analysi s o f

poly(A) + 

### hepati c RN

A wit h 32 

### P

labele da 2u 

### -globuli n cDNA suggestedtha t gasoline treatmen t

di dno t alter significantl

y th e conten t

o f hepati c mRNA codin g fo

r a 2u 

### globuli n (Fig

. 3)

. Immunocytochemical

Localization of

a 2u 

### Globulin In kidney section s fro

msaline-treate drats , staine dspecificall

y fo r a 2u 

### -globulin , many PC T wer e staine dpositively (Fig

. 1B)

. I ngen eral , man y hyalin e droplet s whichstaine d positivel y withMallory' s Heidenhai n metho dals o staine dintensely fo r a 2u 

### -globu

li n (Figs

. IA an dIB)

. However , thi s correla

tio n wa s no t

perfect an dth e obvers e was no tP. 2

a, u -GLOBULI N IN GASOLIN E NEPHROPATH Y 49 

### 1984)

. Th e single exceptio n i s that chroni c in

halatio n o f

gasoline vapor s i s associate dwit h th e appearanc e of

foc i of cell regeneratio n (Buse y an dCockrell , 1984 ; Haider et al.. 1984) , wherea s tubular

regeneratio n di dno t accompan y th e treatmen t reporte dhere

. Evidenc e o f

cytotoxicity , i.e.

. exfoliate depi

thelial cell s an dproteinaceou s casts , were , however , observe dafte

r 9 day s of treatment
. Time-cours e studie s o f

th e toxicit

y o f 2,2.4

- trimethylpentane , Decalin , an disoparaffini c solvent sho w tha t

th e earlies t

alteratio n in kidney structur e i s accumulatio n o f hyalin e material inepithelial cell s ofth e PCT , whic h occur s withi n 1- 5 day s (Alden et al.. 1984 ; Phillip s an dCockrell

. 1984 ; Short et al.. 1986)

. Preliminar

y result s fromou r

studie s indicate tha t

gasoline produce s hyalin e drop

let nephropath y i nasimila

r period ; maximal drople t accumulatio noccurre dby 9 day s o f treatment
. Th e distributio n of

gasoline-induce drenal lesion s an dlocalize daccumulatio n o f hyalin e material suggest s strongly tha t th e primary site o f toxi c actio ni s th e PC T epithelium

. In

formatio no n urin e chemistry gaine dfrom studie s of othe r nephrotoxi c hydrocarbo n mixture s support s thi s conclusion

. Specifi

cally , subacute inhalatio n of isoparaffini c sol

vent increase durinary glucos e an dLD H con centration s an dth e numbe r of epithelial cell cast s (Phillip s an dCockrell

. 1984 ; Phillip s an dEgan , 1984 ; Via

u et al.. 1986)

. ^-mi

croglobuli nexcretio n was unaffectedb y thi s treatmen t suggesting tha t

th e protei n resorp tio n functio n ofth e PC T was intac t (Via u et al.. 1986)

. However , mil dalbuminuria , a n indicato r of glomerula

r damage , ha s been re porte dafte

r inhalatio nof

isoparaffini c sol

vent (Phillip s an dCockrell , 1984 ; Via u et al.. 1986)

. Thus , subtl e alteratio n o f glomerula

r functio n canno t

b e rule dou t

a s contributory t o th e alteratio n of

PC T morpholog y which accompanie s volatil e hydrocarbo n exposure . Hyalin e droplet s are aconstitutiv e featur e o f th e renal PC T epitheliu m(Trum p et al.. 1984) an dare mos t

numerou s in th e S 2 seg ment o f th e PCT , indicatin g th e importanc e of thi s tubul e segment

in protei n resorptio n (Straus , 1964)

. Durin g 2.2.4-trimethylpen

tan e intoxicatio n o f

mal e rat s th e S 2 segment accumulate s hyalin e material to agreate r ex ten t tha n othe r

segment s ofth e PC T (Shor t et al.. 1986)

. Accumulatio n of hyalin e droplet s occur s i ncircumstance s othe r tha n hydrocar

bo ntoxicity , e.g. , durin g heterophag y result

ing fro madministratio nof

nontoxi c low-mo

lecular-weigh

t protein s (Ericsson , 1965)

. Identificatio n ofth e gasoline-induce dhyalin e droplet s a s secondar

y lysosome s was made upot i electro n microscopi c examinatio no f th e PC T epitheliu mo f

gasoline-treate dmal e rat s (Garg et

al.. 1986)

. Qualitativ e an dquan titativ e alteration s o f

lysosome s hav e als o been reporte din studie s of Decalinan diso

paraffini c solvent

toxicit y (Alden et

al.. 1984 : Phillip s an dCockrell , 1984)

. Positiv e identi

fication ofthes e subcellula r structure s a s sec ondary lysosome s was mad e o n th e basi s o f aci dphosphatas e activit

y (Phillip s an dCock

rell , 1984)

. Whether

lysosoma l accumulatio n indicate s atoxi c effec

t o f hydrocarbon s spe cific t o th e lysosome o r

reflects amore generalize ddisturbanc e o f

cellula r metabolis mi s unknown

. Th e compositio n of

renal hyalin e droplet s accumulate ddurin g volatil e hydrocarbo nex posure i s o f

grea t interest

. I

t ha s beenreasone dtha t

i f a 2u 

### -globuli n i s th e sole protei n accumulate dan di

f aspecific defect

ino 2u 

### globuli n metabolis mlead s t o hyalin e drople t nephropath y the nhydrocarbo nexposure present s littl e risk

t o othe r specie s (Alden et al., 1984)

. Ou r

results , however , indicat e that protein s whichare no t

immunologicall y rec ognize da s a 2u 

### -globuli n compos e som e ofth e hyalin e droplet s accumulate dafte r gasoline exposure . Tw o publication s hav e indicate d previously tha t

mal e rat s accumulate a 2u 

### globuli n an dhyalin e droplet s in th e renal cortex durin g intoxicatio nwit hhydrocarbon s othe r tha ngasolin e (Aldenet

ai. 1984 ; Via

u et ai. 1986)

. However , neither o f thes e re port s demonstrate dth e localizatio no f a 2u 

### globulin't o hyalin e droplets

. Further , th e temporal relationshi p between development of hyalin e drople t

nephropathy an daccumu latio n of a 2u 

### -globuli n i s presently uncertain

.P. 3

a 2u -GLOBULI N I N GASOLIN E NEPHROPATH Y 

### 51 inhalatio n o f

unleade d gasoiine . I

n Renal Effects of

Petroleum Hydrocarbons (M . A . Mehlman . C . P. Hem street . III . J. J . Thorpe , an d N . K . Weaver . Eds.) , pp . 57-64 . Princeto n Scientifi cPub. . Inc. . Princeton . NJ . ERICSSON , J . L . E . (1965) . Transportan d digestio n o f he moglobi n i n th e proxima l tubule . II . Electro n micros copy . Lab. Invest. 14.16-25 . FARROW , M. . MCCARROLL . N. . CORTINA . T. . DRAVS . M. . MUNSON , A. . STEINBERG . M. . KJRWIN . C . AN D THOMAS . W . (1983) . In vitro mutagenicit y an d geno toxicit y o f

fuel s an d paraffinichydrocarbon s i n th e Ames' , siste rchromati d exchang e an d mous e lym phom aassays . Toxicologist 3,36 . [Abstract ] GARG . B . D. . OLSON . M . J. . SARKAR . F . H. . AN D ROY , A . K. (1986) . Rena l hyali n drople tan d a 2ll -globuli n accumulatio n aftergasolin e administratio n t o mal e rats . Pharmacologist

28,210 . [Abstract ] HALDER . C . A. . WARNE . T . M. . AN D HATOUM , N . S . (1984) . Rena l toxicity , o f gasolin e an d relate d petro leu m naphtha s i n mal e rats. I n Renal Effects of

Petroleum Hydrocarbons (M . A . Mehlman , C . P . Hem street , III . J . J . Thorpe , an d N . K . Weaver . Eds.) , pp . 73-88 . Princeto n Scientifi cPub. . Inc. . Princeton . NJ . Hsu , S . M. , RAINE . L. , AN D FANGER . H . (1981) . A com parativ e stud y o f

th e peroxidase-anti-peroxidas e metho d an d a n avidin-bioti n comple x metho d fo r studyin g polypeptid e hormone s with radioimmunoas sa y antibody . Amer. J. Clin. Pathol. 75 , 734-738 . KLOSS , M . W. , AN D BUS . J . S . (1985) . Hydrocarbon-me diate d nephrotoxicity . CUT Act. 5,1-8 . KLOSS . M . W. , COX . M . G. . NORTON , R . M. . AN D BUS , J . S . (1986) . Evidenc e o f [ ,4 C-4.5]-2.2.4-trimethylpen tan e bindin g i n urin e an d seru m o f F-344 rats . Toxicologist 6,172 . (Abstract ] KLOSS . M . W. . COX , M . G. . NORTON . R. M. . SWEN BERG , J . A. , AN D Bus . J . S . (1985) . Sex-dependen t difference s i n th e dispositio n o f ( 14 C]-2,2.4-trimethyl pentan e i n Fische r34 4 rats . In Renal Heterogeneity and Target Cell Toxiciiy (P. Bac

h an d E . Lock , Eds.) , pp . 489-492 . Wiley , Sussex . UK . LOURY , D . J. , SMITH-OLIVER . T. , STROM . S. , JIRTLE . R. . MICHALOPOULOS . G. , AN D BUTTERWORTH . B . E . (1986) . Assessmen to f

unschedule d an d replicativ e DN A synthesi s i n hepatocyte s treate d in vivo an d in vitro with unleade d gasolin e o r2.2.4-trimethylpen tane . Toxicol. Appl. Pharmacol. 85 . 11-23 . LOWRY , O . H. . ROSEBROUGH . N . J. . FARR . A . L. . AN D RANDALL . R . J . (1951) . Protei n measurement s with th e Foli n pheno l reagent . J. Biol. Chem. 193,265-275 . MACFARLAND , H . N . (1984) . Xenobioti cinduce d kid ne y lesions : Hydrocarbons . Th e 90-da y an d 2-yea r studies . I n Renal Effects of

Petroleum Hydrocarbons (M . A . Mehlman . C P. Hemstreet . III . J . J . Thorpe , an d N . K . Weaver . Eds.) , pp . 51-56 . Princeto n Scien tifi cPub. , Inc. . Princeton . NJ . MACFARLAND . H . N „ ULRICH . C . E. . HOLDSWORTH . C . E. . KITCHEN , p . N. . HALLIWELL , N . H. . AN D BLUM . S . C . (1984) . A chroni cinhalatio n stud y wit h unleade d gasoline . J. Amer. Coll. Toxicol. 3 . 231-248 . PHILLIPS . R . D. . AN D COCKRELL . B . Y . (1984) . Effect s o f certai n ligh thydrocarbon s o n kidne y functio n an d structur e i n mal e rats . I n Renal Effects of

Petroleum Hydrocarbons (M . A . Mehlman . C P. Hemstreet . III . J . J . Thorpe , an d N . K . Weaver . Eds.) , pp . 89-105 . Princeto n Scientifi cPub. . Inc. . Princeton . NJ . PHILLIPS , R . D* . AN D EGAN . G . F . (1984) . Effec to f C, 0 - C , i isoparaffini csolven to n kidne y functio n i n Fische r 34 4 rats durin g eigh tweek s o f inhalation . Toxicol. Appl. Pharmacol. 73,500-510 . RICHARDSON , K. A. . WILMER , J . L . SMITH-SIMPSON . D. , AN D SKOPEK , T . R. (1986) . Assessmen to f th e ge notoxi cpotentia l o f

unleade d gasolin e an d 2.2.4-tn methylpentan e i n huma n lymphoblast s i n vitro. Toxicol. Appi. Pharmacol. 82 , 316-322 . ROY . A . K. (1977) . Earl y event s i n th e steroidal regula tio n o f a 2u -globuli n i n th e ratliver . Eur. J. Biochem. 73,537-543 . ROY , A . K. . CHATTERJEE . B. . DEMYAN . W . F. . NATH . T . S. . AN D MOTWANI , N . M . (1982) . Pretranslational regulatio n o f

a 2 „-globuli n i n ratlive rby growt h hor mone . J. Biol. Chem. 257,7834-7838 . ROY , A . K. , AN D NEUHAUS . O . W . (1966) . Identificatio n o f raturinary protein s b y zon e an d immunoelectro phoresis . Proc. Soc. Exp. Biol. Med. 1,894-899 . SARKAR . F . H. . MANCINI . M . A. . NAG , A.C , AN D ROY . A . K. (1986) . Cellula rinteraction s i n th e hormona l in ductio n o f

o 2 „-globuli n i n ratliver . / . Endocrinol. Ill , 205-210 . SHORT , B. . BURNETT , V. . COX . M. . BUS . J. , AN D SWEN BERG . J . (1986) . Dos e response studie s o f 2.2.4-tri methylpentane(TMP)-induce d nephrotoxicity an d re tentio n i n th e mal e rat. Toxicologist 6 , 172 . [Abstract ] STRAUS , W . (1964) . Cytochemica l observation s o n th e relationshi p betwee n lysosome s an d phagosome s i n kidne y an d live rby combine d stainin g foraci d phos phatas e an d intravenousl y injecte d horseradis h peroxi dase . / Cell Biol. 20,497-507 . . THOMAS . P . S . (1980) . Hybridizatio n o f denature d RN A an d smal l DN A fragments transferrred t o nitrocellu lose . Proc. Sail. Acad. Sci USA 77 , 5201-5205 . TRUMP , B . F. , JONES , T . W. . AN D HEATFIELD . B . M . (1984) . Th e biolog y ofth e kidney . I n Renal Effects of Petroleum Hydrocarbons (M . A . Mehlman . C . P . Hemstreet . III . J . J . Thorpe , an d N . K . Weaver . Eds.) , pp . 27-49 . Princeto n Scientifi cPub. . Inc. . Princeton . NJ . VIAU . C . BERNARD , A. . GUERET . F. . MALDAUGLE , P. . GENGOUX . P. , AN D LAUWERYS . R. (1986) . Isoparaffinicsolvent-induce d nephrotoxicity i n th e rat . Toxicology 38 , 227-240 .P. 4



# t^NA L Hmi N DROPLE T ACXWUlAfld N AFTE R GASOLIN E AD^N^TRATIO N T

O MAL E RAT

S
"yx^y&'^J&jgy*-' 

# .W^^qrr-^A-^ -

^ej^c e Department

•yy^' r "•

MA YB E DISTRIBUTE D OUTSID E GENERA L MOTOR S No t t o b e Reproduce d Withou t Permissio nP. 5



### Rena l Hyali n Drople t an d a „ -globuli n Accumulatio n afte r Gasolin e Administratio n t o Mal e Rat s

B.D . Gar g an d M.J . Olso n Biomedica l Scienc e Departmen t Genera l Motor s Researc h Laboratorie s

Warren , M I 4809 0

F.H . Sarka r an d A.K . Ro y Biologica l Science s Departmen t Oaklan d Universit y Rochester , M I 4806 3P. 6



### Abstrac t
Saturate d branched-chai n aliphati c hydrocarbons , suc h a s thos e foun d i n moto r fuels , induc e nephrotoxicit yi n mal e rats . Treatmen t o f mal e rat s wit h unleade d gasolin e (0. 2 - 2. 0 mL/k g bod yweight , p.o. ) fo r nin e day s cause d a marke d increas e i n th e numbe r an d siz e o f hyali n (protei n resorption ) droplet s i n epithelia l cell s o f th e proxima l convolute d tubule s (PCT ) an d increase d cellula r exfoliatio n a t hig h dos e levels
. N o othe r treatment-relate d pathologica l effect s wer e observe d i n th e glomeruli , dista l tubule s o r medulla
. Transmissio n electro n microscop yo f kidney s showe d tha t gasolin e cause d a strikin g increas e i n th e numbe r an d siz e o f phagolysosome s i n PC T epithelia l cells
. Th e rena l conten t o f ce

_-globulin , a majo r urinar yprotei n o f mal e rats , wa s increase d 3-fol d afte r gasolin e administratio n (0. 2 mL/kg , p.o. , 9 days) ; n o furthe r increas e wa s observe d a t highe r doses
. Th e accumulatio n o f a „ -globulin , measure d b

yradioimmunoassa y(RIA ) , wa s reverse d withi n thre e day s o f terminatin g gasolin e administration
. Immunoperoxidas e stainin g o f kidne ytissu e section s fo r a „ -globuli n reveale d increase d quantitie s o f th e protei n i n treate d rat s an d larg e accumulation s o f antige n localize d i n PC T epithelia l cell s whic h containe d hyali n droplets
. Th e hepati c concentratio n o f a . - globuli n an d th e quantit yo f a„ -globuli n mRN Ai n live r wer e no t altere d b ygasoline
. Thes e aat a sho wtha t a~ -globuli n i s accumulate d i n th e kidney s o f gasoline-intoxicate a mal e rat s an d sequestere d specificall yi n som e o f th e hyali n droplet s characteristi c o f gasoline-induce d nephropathy
. Ahydrocarbon

induce d defec t i n th e rena l lysosoma l degradatio n o f lo w molecula r weigh t proteins , rathe r tha n increase d synthesi s o f thes e proteins , appear s t o caus e hyali n drople t accumulation
.
Introductio n Toxicologica l assessmen t o f volatil e petroleu mhydrocarbo n mixture s use d a s moto r fuels , solvents , pain t an d varnis h additives , degreasin g agent s an d industria l intermediate s ha s reveale d dose-dependen t mil d alteration s i n th e for man d functio n o f th e rena l corte x followin g subacut e an d chroni c exposur e (1)
. Th e importanc e o f understandin g hydrocarbon-mediate d nephropath y wa s increase d greatl ybyth e observatio n tha t rat s expose d fo r 1 8 o r mor e month s t o wholl yvolatilize d unleade d gasolin e exhibite d a n increase d incidenc e o f rena l carcinom a (2)
. Th e occurrenc e o f tumor s wa s sex - an d species-specific ; mal e rat s onl yexhibite d treatment-relate d rena l neoplasia
. Analysi s o f gasolin e an d othe r comple x hydrocarbo n mixture s i n short-ter massay s fo r mutagenesi s ha s produce d equivoca l o r negativ e result s (3)
. Thus , researc h ha s bee n directe d towar d elucidatin g th e mechanism s bywhic h thes e agent s induc e over t nephrotoxicit yi n th e hop e tha t a n epigeneti c even t relate d t o toxicity , suc h a s a n increas e i n th e rat e o f cel l turnover , ma yexplai n th e occurrenc e o f rena l tumors .
- 1

-P. 7



## collidine buffe r (p H 7.4) . Seria l section s o f kidne y wer e staine

d wit hMallory' s Heidenhai nmetho

dor immunostaine d fo r art-globuli nwit hthe avidin-bioti nprocedur e (5) . Briefly , th e tissu

e wa s deparaffinized , rehydrated an dincubatedi na solutio no f 3% norma l goa t serum , 3% bovine seru m an d2% gelatin . Section s wer e thenincubated wit hpolyclona l anti-o „ -globuli n(rabbi t IgG ) for 3 0 mi nan

d washed wit hphosphat e buffered saline (PBS ; 0.1 4 M NaCI , 0.0 1 M

Na-phosphate , pH 7.4) . The section s wer e furthe r incubate

d for 3 0 mi nwit hbiotinylatedanti-rabbi t Ig G(goat IgG ) an dwashed . A

fina l 3 0 mi nincubatio nwit hfreshl y

prepared avidin-bioti nreagen t (Vecto r Laboratories , Burlingame , CA ) wa s followed b

y extensiv

e washing i nPBS . The tissue s wer e then place di n0.0 5 M

Tris-HCl , p

H 7.0 , containin

g 0.001 4 M

3,3' - diamino-benzidine tetrahydrochloride an d 0.00 3 M

hydroge n peroxide fo

r 1 5 min . Th

e reactio

nwa s terminatedwit ha 1 5 mi n rins e i nrunnin

g wate r an

d th e section s wer e counterstainedwit h Harri s hematoxylin . Dot-blo t assa

y o f a„ -globuli nmRN A inliver Tota l hepati c RNA , extracte dwit hpheno l an d sodiu m dodecy l sulfat e wa s use

d a s th

e startin

g materia l t o isolat e pol y (A) - containin

g RN A b y oligo(dt)-cellulos e chromatography . Seriall y dilutedpoly(A)-containin g RNA o wa s spotte

do nnitro-cellulos e filter s an

d hybridized .wit

hP-labele

d a „ -globuli ncDN A (6) . Afte r hybridization , quantitativ e bindin

g - u 

## o f th e probe wa s determined b

y autoradiograph

y an d densitometry . Radioimmunoassa

y o f g p -globuli ni

nkidne y an d live r Homogenate s o f whol e kidne y an d live r wer e prepared an d centrifuged a t 100,00 0 xg ; th e supernatan t flui dwa s used for doubl e antibod

y RI A o f a „ -globuli na s describedb y Ro y (7) . The concentratio

no f a . -globuli ninth e supernatan t fractio nwa s expressed a s /i

g a „ -globuli nper m g tota l protein .

-3 -P. 8

/ ! *

! ' i f; n r o IP. 9 

# \\t&±'*%&&*"< i-V&J-

/ * F'***? ° a* \ • V * " 

# ?-'" . .;* $ j i



# i

^»-- v ^ ^ T ? ^ ••*:> >

!• ' . p; u iP.1 0

• * *'V - « fj* f i " • r v .

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. v.-.?* ' » -*»* " - •SS.'fv * *.,•;.>' . ;*(•-. .. . ... ; .t" -


# j g f * *» - -" ; 

# s&V'.¥*> * zm**i;.:fr

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### * -*.--•*•*": ; •• ! 

### ^iv^^jfe^5^^:^ .

i * ' 

# •z~%ftmz':

..••'.,... . i, ' a"•...'.*;.•;<<*Kv- r_r< - "c?r;'<" , 

### -.v . ' ' • ^'.** . A-*.^? M ' .* • •• - T^.;**** ^ HmJy 

### k^P>.r*'-* J 

### '--^-^ i

Figur e 3
- 7

-P.1 1



## Tabl e 1

Summar yo f Hydrocarbo n Type s i n Unleade d Gasolin e

Hydrocarbo n Carbo n skeleto n Isomer s Weigh t % Clas s Lengt h Detecte d

n-paraffin s C „ - C. Q 8 13. 0 isoparaffin s C - C g 

### 3 3 28. 2 napthene s C f i 

### - C „ 1 1 3. 6
olefin s C 3 

### - C. g 2 0 5. 5
aromatic s C g 

### - C 1 0 

### 3 3 36. 6

Total s 10 5 86. 9
Liqui d gasolin e wa s analyze d b ycapillar ycolum n ga s chromatograph y(Hewlett-Packar d mode l 5880A , F.I.D. , 5 0 mx 0. 2 mmfuse d silic a colum n coate d wit h cross-linke d dimethylsiloxan e gum , ove n temperatur e programme d fro m0 t o 20 0 C , injecto r and

detecto r a t 20 0 an d 25 0 C , respectively)
. Typica l result s fro m analyse s o f thre e lot s o f gasoline
.

-9

-P.1 2



### Tabl e 3

Effec t o f Recover yTim e o n Rena l a „ -globuli n Conten t Afte r Gasolin e Administratio n

Treatmen t Rena l a ~ -globuli n (/xg/m g tota l protein )

Saline , 2. 0 mL/k g x 9 d Gasoline , 2. 0 mL/k g x 9 d Gasolin e x 9 d + 3 d recover y Gasolin e x 9 d + 6 d recover y Gasolin e x 9 d + 9 d recover y Gasolin e x 9 d + 1 2 d recover y 24. 2 ± 4. 2 (3 ) 127. 0 ± 11. 0 (6 ) 33. 0 * 5. 1 31. 9 26. 2 ±4. 1 34. 0 ±5. 2 ( 3 )

( 2 ) ( 3 ) ( 3 ) * *

1 >t c if c 

### mea n * S.E.M. , n i n parentheses
. differen t fro mal l othe r grou p means , p=0.0 1 (Duncan' s test)
. Rat s wer e administere d salin e o r 2. 0 mL/k g gasolin e bygavag e onc e dail yfo r nin e consecutiv e day s followe d b yth e recover yperiod s shown
.

-11

-

### Reference s

1
. Rena l Effect s o f Petroleu mHydrocarbons , M.A . Mehlman , G.P . Hemstreet , III , J.J . Thorp e an d N.K . Weaver , eds. , Princeto n Scientifi c Publishers , Princeton , NJ , 1984
. 2
. MacFarland , H.N. , CE . Ulrich , CE . Holdsworth , D.N . Kitchen , N.H . Halliwel l an d S.C . Blum , J . Am . Coll
. Toxicol
. 3 : 231

- 248 , 1984
. 3
. Loury , D.J . an d B.E . Butterworth , C.I.I.T . Activitie s 6 : 1-7 , 1986
. 4
. Roy , A.K . an d O.W . Neuhaus , Proc . Soc . Exp . Biol
. Med
. 1 : 894-899 , 1984
. 5 . Hsu , S.M. , L . Rain e an d H . Fanger , Am . J . Clin
. Path
. 75 : 734-738 , 1981
. 6
. Roy , A.K. , B . Chatterjee , W.F . Demyan , T.S . Nat h an d N.M . Motwani , J . Biol
. Chem
. 257 : 7834-7838 , 1982
. 7 . Roy , A.K. , Eur . J . Biochem
. 73 : 537-543 , 1977
.

-13

-P.1 4



# GMR-571 6 RAPI D POST-EXPOSUR E DECA Y O F GASOLINE-INDUCE D HYALI N DROPLET

S AN D ^-GLOBULI N ACCUMULATE D I N TH E MAL E RA TKIDNE Y Bhagwa n D . Garg Michae l J . Olso n Biomedica l Scienc

e Departmen t Fl"Danya n land-Ohiversit

y Februar y 11,198 7 

# iboratoi i Warren , fclichigan 4809 0

MA YB E DISTRIBUTE D 0UTS10 E GENERA L MOTOR S Not t o b e Reproduce d Withou t Permissio nP.1 5

GMR-571 6

Rapi dPost-Exposur e Deca y o f Gasoline-Induce dHyali nDroplet s an da „ -Globuli nAccumulate di nth e Male Ra t Kidne y

Bhagwa nD . Gar g an dMichae l J . Olso n Biomedica l Scienc e Departmen t Genera l Motor s Researc hLaboratorie s Warren , Michiga n48090-905 8

William

F . Demya nan

dAru nK . Ro y Departmen t o f Biologica l Science s

Oaklan dUniversit y Rochester , M I 4806 3

Presente da t the 26t hAnnua l Meetin g o f th e

Societ y

o f Toxicology Washington , D.C . Februar y

26 , 198 7whic haccumulate s hyali ndroplet s subsequentl y shoo s increasedlevel s o f DN A replication . Interes t i

nthe potentiall y

advers e healt heffect s o f gasolin e le du s t o develo pa short-terra mode l fo r examinin g th ©toxicit y

b f gasolin e i

nrat s (5) . Wor k a t G

M Researc hLaboratorie s ha s shoe m tha t a majo r componen t o f gasoline indueod hyali ndroplet s i s th e androgen-dependen t ra t urinary protei ne„ - globulin . Accumulatio no f thi s protei ndu e t o defectiv e rena l (lysosomal degradatio ni s propose da s a nintegra l even t o ngasol5ne=5nduce d cel l deat h (5,8) . H ©hav e attempte d t o understandi f an dho w gasoline alter s the aetab©15s ®o f e * -globuli nan dwhethe r suc halteratio ndoes , i nfact , contribut e t o the Nephropath y associate dwit hfue l hydrocarbon , exposure . Thi s repor t describe s th e relationship s betwee nduratio no f gasolin e administratio n o r dose o f gasoline an dth e rena l accumulatio no f e « -globulin . Further , th e rat e o f decay

o f hyali ndroplet s andaccumulate de „ -gfobuli nafte r terminatio n o f gasoline exposur e wa s determined .

MATERIAL S AN

D METHOD S
Experimenta l animal s an dchemical s

D Mal e Fische r 34 4 rat s (CDF (F-344)/Cr l BR ; Charle s Rive r Laboratories , Wilmington , MA ) weighin g approximatel y 25 0 g wer e use dthroughou t thes e studies . Rat s were maintaine d i ncondition s consisten t wit hAAALA C recofftmendations wit hPurin a 501 2 chow an dta pwate r freel y availabl e durin g th e entir e period . Fo r dose-respons e an dtime-cours e experiment s rat s wer e administere dphysiologica l saline , cor noi l o r a commerciall y availabl e unleade dgasolin e (nea t o r mixe dwit hcor noil ) b y intragastri c intubatio nonc e daily . I nhyali ndrople t decay studie s rat s wer e treate dwit hgasolin e (2. 0 mL/kg , p.o. , 9 days ) o r gasolin e plu s estradio l ( 1 mg/k g i na propylen e glyco l vehicl e (7) , s.e , 4 days , startin g o nth e fina l day o f gasolin e treatment) . Followin g conjoin t gasolin e an dhormon e treatment , animal s wer e seriall y sacrifice da t 3 , 6 an d9 day s afte r th e las t dos e o f gasoline . Control s receivin g salin e an drat s treate dwit hgasoline alon e wer e als o allowe dt o recove r fo r 3 t o 9 day s afte r terminatio no f treatment . Radioimmunoassa y

o f a ^ -globuli ni nkidney

an dlive r Th o cytosoli c fractio no f kidney an dlive r homogenate s wer e prepare dfro m sample s o f th e perfuse dorgan s (se e below ) an dassaye dfo r G

_-globuli nb

y double-antibody

radioimmunoassa y a s describe d b

y Ro y (8) . The quantit y o f fi- - globuli ni

nthe cytosoli c fractio nwa s expresse drelativ e t o tota l protei n whic hwa s determine db y th e metho do f Lowry e t al . (9) . Histologica l procedure s Eightee nhour s afte r th e fina l dos e o f gasolin e o r afte r th e perio do f recovery , rat s were anesthetize dwit hanintraperitonea l injectio no f pentobarbital , perfuse dvi a th e hear t wit hcol d0.9 % sodiu m chloride solutio n an dthe kidney s an

dliver s were removed , fixe db y immersioni

n10 % neutra l buffere dformalin , processe droutinel y an dembedde di

nparaffin . Section s o f paraffin-embedde d kidney ( 4 fim

thickness ) wer e staine dwit hMallory' s Heidenhai nmetho dfo r visualizatio no f hyali ndroplets .
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LU 

# o cc Q . O ) 

## E
*•» » O ) 3 

### Z ) C D 

## O MO C D 70 - I 3



# \fo g 

## KIDNE Y

1 2 1 5 

## TIM E (DAYS ) 1 8

Figure 1 . Effec t o f recover y tim e an destradio l treatmen t afte r terminatio nO T gasolin e exposur e onrena l a-, -globuli ncontent . Rat s wer e treate d wit hvehicl e alone (X) , 2. 0 mLfkg gasoline , p.o . fo r nin e day s (# ) o r gasolin e plu s estradio l ( 1 mg/kg , s.e. ) fo r fou r day s beginnin g o n the fina l day

o f gasolin e administratio n(O) • Dat a are expresse da s mea n* SE M

a~-globuli nconten t i nlive r an

dkidney . Arro w

indicate s terminatio no¥gasolin e treatmen t o f rats . Significantl y differen t fro m al l othe r groupmeans , P < 0.0 1 (Duncan' s test) . Differen t grou pno t receivin g estradiol , P < 0.05 , fro m correspondin g valu e fo r * * P. < 0.0 1 (Duncan' s test) .



### -5 -? 

## ~i*f*»&t*L



# *'£V£y&&: ' ..

. *v . iM^fi^><-' .

Figur e 2
. Photomicrograph s o f rena l tissu e section s fro msaline-treate d (2. 0

mL/kg , p.o. , 9 days ; Pane l A ) and gasoline-treate d (2. 0 mL/kg , p.o. , 9 days ; Pane l B ) rats . Enhance d hyali n droplet (-^-. ) accumulation , correlate d t o a n increas e in a ? -globuli n content , i s obviou s i n th e kidne yo f th e gasoline-treate d anima l compare d t o control
. Mallory' s Heidenhai n stain
. 50X
. 

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-P.1 9

Figure4 . Effec t o f 9 day s post-gasolin e exposur e (2. 0 mL/kg , p.o. , 9 days )

recover y

onhyali ndrople t numbe r ( -^^ - ) i nrena l PC T epithelia l cells . Compar e t o Figs . 2 an d3 . Mallory' s stain . 50X .



### -9 -P.2 0

SUMMAR YAN D CONCLUSION S

A short-term

ora l dosin g paradig m wa s employe dt o stud y th e rat e d f accumulatio nanddisappearanc e o f a „ -globulin , a protei nintimatel y associate d wit hthe nephrotoxicit y o f fue l hydrocarbon s i nmale rats , i

nth e kidney

durin g gasolin e administration . Th e result s o f specifi c experiment s show that ;

1 . Th e hepati c conten t o f a „ -globuli nwa s no t significantl y affecte db

y gasoline administratio nwhe nevaluate da t a variet y o f gasolin e dose s an d duration s o f exposure . 2 . Th e rena l accumulatio no f a

_-globuli ninduced b y gasolin e wa s rapidl y reversibl e afte r terminatin g subacut e administratio no f gasoline ; complet e reversa l o f rena l a « -globuli naccumulatio noccurre dwithi nthre e day s o f discontinuin g gasoline treatment . However , resolutio no f hyali ndrople t nephropath

y require d 6 t o 9 day s post-exposur e recovery . 3 . Inhibitio no f the hepati c synthesi s o f a „ -globuli nimmediatel y prio r t o terminatio no f gasolin e administratio nenhance dthe remova l o f a „ -globuli n accumulate di

nthe kidney . Thi s suggest s tha t hydrocarbon-induce d rena l accumulatio no f a « -globuli ni s directl y dependen t o nth e rat e o f hepati c synthesi s o f a„ -globulin . I nsummary , thes e dat a sugges t tha t unleade dgasolin e induce s a nephrotoxi c respons e i

nmal e rat s characterize d b y rena l accumulatio no f a « -globulin . Th e mechanis m

fo r suc ha neffec t i s unknown . However , dat a presente dar e consisten t wit hth e hypothesi s tha t gasolin e produce s a transient , readil y reversibl e diminutio no f the rena l catabolis m (degradation ) o f lo w molecula r weigh t proteins , suc ha s a „ -globulin .



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