# OAR Box 1830

Prepped by Ollie Stewart



## Document

Number: 

# 3)IV-A-1 0 Docket Number: 

# A-87-1 1

Printed 11/13/20087:20:50AM 

### Sheet 3 of 232 P. 1w- , •• ••<w>r«|>«W!.'|ay W y. f ,. - , Taje^ T

EPA/625/3-91/019 F Septembe r199 1 

## ALPHAS-GLOBULIN : ASSOCIATIO N WIT H CHEMICALLYINDUCE D RENA L TOXICIT YAN D NEOPLASI A I N TH E MAL E RA T

Prapara d forth a Rla k Aaaaaaman t Foru m U.S . Envlronmanta l Protactlo n Agancy

Waahlngton , D.C . Principa l Authora Kai l P . Baatcka , Ph.D . Gordo n C . Hard , Ph.D . D.Sc . Letiti a M . Tahan . M.S .

Imogan a Savi n Rodgara , Ph.O . Robar t E . McGaughy . Ph.D . Tachnlca l Pana l Kar i P . Baatcka . Ph.D. . Co-Chai r Latili a M . Tahan . M.S. . Co-Chai r Marlo n P . Copley . O.V.M. . JuH a Du , Ph.D . Robar t E . McGaughy. Ph.D . Willia m E . Papain , Ph.D . Chary l Slege l Scott . M.S . Lawranc a R . Valcovlc , Ph.D . Rla k Aaaaaaman t Foru m Staf f Exaeutiv a Dlractor : Doroth

yE . Patton , Ph.D . J.D . Seianc a Coordinator : Imogana Savi n Rodgara , Ph.D . Consultant : Gordo n C . Hard , B.V.Sc. , Ph.D. . D.Sc. , F.R.C . Path. . F.R.C.V.S. . F.A. Tox . Sol .



## Ris k Assessmen

t Foru m U.S . Environmenta l Protectio n Agenc y Washington , DC 20460

X& X„ .

## Disclaime r

Thi s documen t ha s bee n reviewe d i n accordanc e wit h U.S . Environmenta l Protectio n Agencypolic yan d approve d fo rpublication . Mentio n o ftrad e name s o r commercia l product s doe s not constitut e endorsemen t o rrecommendatio n fo ruse . 

## Content s

Lis t o fTable s v i List o fFigure s vi i List o fAbbreviation s vi i Externa l Pee rReviewer s i x Prefac e xi i I . Executive Summary1 II . Introductio n 6 Par t 1-Nephrotoxlclt y III . Hyalin e Droplet s an d Alphas-globulin ; Physiolog yan d Biochemistr

y1 1 A . Filtration . Reabsorption , an d Catabolis m o f Low-Molecular-Weigh t Protein s byth e Kidney1 1 B . Hyalin e Droplet s i n Rena l Tubule s 1 3 C . Factor s Affectin g KidneyAccumulatio n o f Low-Molecular-Weigh t Protein s 1 4 D . Th e Alphas-globuli n Superiamil y o fProtein s 1 5 E . Characteristic s ofAlphas-globuli n 1 9 F . Se xan d Specie s Compariso n ofUrinaryProtei n Conten t o

fth e Upocali n Superfamil y 2 0 G . Nonoovalen t Bindin g t o Alphas-globuli n an d it s Homobgue s 2 1 1 . Chemica l entitie s boun d t o alphas-globuli n 2 1 2 . Natur e ofth e associatio n 2 2 3 . Bindin g ofCIG A t o othe rmacromolecule s 2 3 4 . Specificityo

fth e interactio n o

fCIG A wit h alphas-globuli n 2 3 H . Catabolis m o

fAlphas-globuli n Complexe d wit h CIG A 2 4 I . Structure-ActivityRelationship s fo rCIG A 2 6 IV . Alphas-globuli n Nephropath y27 A . Pathologi c Feature s o fAlphas-globuli n Nephropath y2 7 B . Rat Urin e Chemistr yan d CIG A 3 0 C . Specie s Variatio n i n th e Rena l Respons e t o CIG A 3 0 D . Factor s Affectin g th e Expressio n o fAlphas-globuli n Nephropath y3 2 1 . Age-relate d effect s 3 2 2 . Effect o

fhormon e manipulatio n 3 3 3 . Geneti c variant s 3 3 4 . Alphas-globuli n infusio n i n femal e rat s 3 3

## Contents (cont. )

E

. Chroni cProgressiv e Nephropath y3 4 F

. Rena l Toxicit yObserve d i n Chroni cBioassay s o f Chemical s tha t Induce d Kidne yTumor s i n Rat s 3 5

Part 2-Carclnogenlclt y V

. Pathologi cFeature s o fRena l Carcinogenesi s Induce d b y Classica l Carcinogen s 4 1 A

. Earf yNephrotoxicit y4 2 B

. Karyomegal y4 2 C

. Tubul e Cel l Hyperplasi a 4 2 D

. Adenom a 4 3 E

. Adenocarcinoma s an d Carcinoma s 4 3 F

. Tumo rProgressio n 4 3 G

. Sit e o fOrigi n o fRena l Tubul e Tumor s 4 4 VI

. Neoplasti can d Preneoplasti cLesion s Observe d i n th e 2-Yea rBioassay s 4 4 A

. Generi cConsideration s 4 4 B

. Rena l Tumo rIncidenc e 5 0 C

. Histogenesi s o fRena l Tumor s 5 0 D

. Rena l Tumo rLatenc yan d Progressio n 5 1 E

. Inductio n o fOthe rTumo rType s 5 1 VII

. Additiona l Evidenc e Concernin g th e Rena l Carcinogenicit yo fCIG A5 2 A

. Geneti cToxicolog yStudie s 5 2 B

. Initiation-Promotio n Sudie s 5 5 VIII

. Compariso n o fCIG Awit h Classica l Rena l Carcinogen s 5 7 IX

. Evidenc e Concernin g Huma n Kidne yCance r5 8 A

. Morpholog yan d Histogenesi s 5 8 B

. Incidenc e an d Mortalit y5 9 C

. Environmenta l an d Lifestyl e Factor s 6 0 D

. Occupationa l Factor s 6 1 E

. Rena l Cance ran d Hydrocarbon

. Solvent , o rPetroleu m Produc t Exposur e 6 1 X

. Evidenc e fo rDose - an d Time-Dependen t Progressio n fro m Earl yt o Lat e Lesion s 6 3 A

. Associatio n Betwee n CIGA , Hyalin e Drople t Formation , an d Alphas-globuli n Accumulatio n 6 4 B

. Associatio n Betwee n Hyalin e Drople t Formation , Cel l Necrosis , an d Tubul e Cel l Regeneratio n 6 5 I V 

## Content s (cont. )

C

. Progressio n t o Cas t Formation , Tubul e Dilation , an d Mineralizatio n 6 8 D

. Associatio n Betwee n CIG Aan d Chroni cProgressiv e Nephropath y7 0 E

. Evidenc e Concernin g Progressio n fro m Nephrotoxicit yt o Rena l Neoplasi a 7 1

Par t 3-Evaluatlo n o fth a Hypothesi a XI

. Summar yo fth e Evidenc e o n th e Rena l Effect s o fCIG A7 3 A

. Associatio n Betwee n Alphas-globuli n Accumulatio n an d Nephrotoxicit y7 3 B

. Associatio n Betwee n Nephrotoxicit yan d Rena l Cance r7 4 C

. Informatio n Reducin g Confidenc e i n th e Conclusio n tha t th e Alphas-globuli n Respons e i s Specifi ct o th e Mal e Ra t 7 5 XII . Conclusion s 7 6 XIII

. Researc h Need s 7 8 Par t 4-Sclenc e Polic y XIV

. Backgroun d an d Introductio n 8 1 XV

. Basi s fo rScienc e Polic yo n Mal e Ra t Kidne yTumor s 8 1 A

. Low-Molecular-Weigh t Protein s i n th e Ra t 8 1 B

. Progressio n fro m Chemicall yInduce d Alphas-globuli n Accumulatio n t o Nephropath yan d Neoplasi a 8 2 1

. Overvie w8 2 2

. Specificit yo fth e sequenc e t o th e mal e ra t 8 3 XVI

. Scienc e Polic yStatemen t 8 5 XVII

. Guidanc e fo rEvaluatin g Chemicall yInduce d Mal e Ra t Rena l Tubul e Tumor s 8 5 A

. Rena l Tubul e Tumor s i n Mal e Rat s an d Alphas-globuli n Accumulatio n 8 6 B

. Additiona l Informatio n Usefu l fo rth e Analysi s 8 7 C

. Us e o fth e Dat a fo rRis k Assessmen t 8 8 XVIII

. Nephropath ya s a Toxi cEndpoin t 8 9 Appendi x- Non-neoplasti cEffect s o fHyalin e Drople t Inducer s 9 0 Reference s 9 9

## List o fTable s Lis t o fFigure s

Numbe rPag e 1

. Example s o forgani cchemical s tha t hav e produce d rena l injuryi n mal e rat s characterize d b yhyalin e drople t accumulatio n bu t no t i n femal e rat s o rothe rspecie s 7 2

. Superfamil yo flipophili cligand-bindin g carrie rprotein s 1 7 3

. Summaryofth e histopatholog yan d lesio n progressio n reporte d i n alphas-globulin-associate d nephrotoxicit y2 8 4

. Summar

yo fth e histopatholog yo fspontaneou s chroni cprogressiv e nephropath yo fagin g rat s 3 5 5

. Summar

yo fdat a o n non-neoplasti can d Pre-Neoplasti ckidne y lesion s i n mal e rat s associate d wit h eigh t mode l compound s tha t induce d rena l tumor s i n 2-yea rbioassay s 3 7 6

. Summar

yo fdat a fro m 2-yea rbioassay s o n non-neoplasti can d pre-neoplasti ckidne ylesion s i n mic e an d femal e rat s expose d t o eigh t mode l compound s tha t induce d rena l tumor s i n mal e rat s 3 8 7

. Incidence s o frena l tubul e preneoplasi a an d neoplasi a i n rat s take n fro m 2-yea rbioassay s o n eigh t mode l substance s 4 5 8

. Incidence s o frena l tubul e preneoplasi a an d neoplasi a i n rat s take n fro m 2-yea rbioassay s o n chlorothaloni l an d trichloroethylen e 4 8 9

. Summar

yo fgenotoxich ydat a fo reigh t selecte d mal e ra t kidne ycarcinogen s 5 3 10

. Summar

yo fgenotoxicit ydat a fo rtw o selecte d non-CIG Amal e ra t kidne ycarcinogen s 5 4 11

. Incidenc e ofmedullarymineralizatio n i n mal e rat s durin g inhalatio n exposur e t o unleade d gasolin e 7 0 Numbe rPag e 1

. Diagra m o fzonatb n an d tubul e segmentatio n i n ra t kidne y1 0 2

. Schemati crepresentatio n o fendocyti cuptak e o ffiltere d protein s 1 2 3

. Schemati crepresentatio n o fth e uptak e an d fat e o fabhaoy-globuli n oomplexe d wit h a CIG Ai n hydrocarbo n nephrotoxicit y2 5 4

. Dose-respons e relationshi p betwee n rena l hyalin e drople t accumulatio n (D ) an d | 3 H]-thymidin e labelin g inde x6 6 5

. Effec t o f0-2,00 0 pp m unleade d gasolin e o n continuou s uptak e o fJ 3 HJ-Td R 6 7 6

. Time-sequenc e fo rth e developmen t o fhyalin e droplets , regeneratin g tubul e epithelium , an d tubul e dilatio n i n mal e F34 4 rat s 6 9

V I VI I

## List o fAbbreviation s

AA T AB S «2u- 9 CH O C I CIG A CP N 1,2-DC B 1.4-DC B DE N DM N EHE N FBP A H 4 E IRD C ML A MT D MU P NA G NB R NT P NC I O R R R P 1 P 2 P 3 SA L SC E SD S SEE R SI R SLR L SM R T K TM P TMPO H UD S aspartat e aminotransferas e

chromosom e aberration s i n CH O cell s abha2u-globuli n Chines e hamste rovary confidenc e interva l £hembal(s ) Inducin g abhsgu-filobuli n Accumulatb n chroni c progressive nephropath y 1,2-dtehbrobenzen e 1,4-dtehbrobenzen e diethylnitrosamin e dimethylnitrosamin e N-ethyl-N-hydroxyethylnitrosamin e N-(4'-fluoro-4-biphenylyl)acetamid e hematoxyli n an d eosi n Intamatbna l Researc h an d Developmen t Corporatb n TK-gen e mutatio n assa yi n L5178 Ycell s maximu m tolerate d dos e mous e majo rurinar

yprotei n N-acetyl-B-glucosaminidas e NC I Btack-Reite rra

t Nationa l Toxicolog yProgra m Nationa l Cance rInstitut e odd s rati o relative ris k first convolute d segmen t ofproxima l tubul e secon d convolute d segmen t o fproxima l tubul e para rect a o

fproxima l tubul e Salmonell a siste rchromati d exchang e sodiu m dodecy l sulfat e Surveillance , Epidemiolog yan d En d Results Progra m o

fNC I standardize d incidenc e rat b sex-linke d recessive letha l standardize d mortalit

yrat b thymidine-kinas e 2,2.4-trimethytpentan e 2.4.4-trlmethylpentano l unschedule d DN A synthesi s 

## Externa l Pee rReviewers

A n interi m draft , prepare d i n Septembe r1990 , wa s evaluate d a t a two-dayPee r ReviewWorksh p sponsore d byth e EP A Ris k Assessmen t Forum . Th e meeting , hel d i n Gaithersburg , Maryland , o n Novembe r1 3 an d 14,1990 , wa s chaire d b yDr . Richar d Griesemer , directo ro fth e Divisio n o

fToxicolog yResearc h an d Testing . Nationa l Toxicolog yProgra m (NTP) . Invite d participant s ar e liste d bebw . Th e Worksho p proceeding s wil l b e publishe d separately . Announcemen t o fthei r availabilit

ywil l b e throug h th e Federa l Register . Pee rRevie wWorksho p - Invite d Participant * Joh n Ashb

y(Cance rWorkgrou p Chair ) Imperia l Chemica l Industrie s Centra l Toxicolog yLaboratory Cheshire . Englan d Debora h Barsott i Divisio n o

fToxicolog y Agencyfo rToxi c Substance s Diseas e Registr

y(ATSDR ) Atlanta , Georgi a R . Danie l Ben z Cente rfo rFoo d Safet y an d Applie d Nutritb n Foo d an d Dru g Administrat e Washington . D.C . Willia m Busey a Experimenta l Patholog y Laboratorie s Hemdon . Virgini a MurrayCon n Healt h Effect s Divisb n Directorat e fo rHealt h Science s Consume rProduct Safet yCommissb n Bethesda , Marylan d Michae l Etwel l Divisb n o

fToxicolog y Researc h an d Testin g NIEH S Researc h Triangl e Park , Nort h Carolin a Sco t L . Eusti s Divisb n o

fToxicolog y Researc h an d Testin g NIEH S Researc h Triangl e Park , Nort h Carolin a Willia m Farlan d USEP A Offic e o fHealt h an d Environmenta l Assessmen t Washington , D.C . Penn yFenne rCris p USEP A Healt h Effect s Divisb n Offic e o fPesticide s Product s Washington , D.C . Richar d Grieseme r(Worksho p Chair ) Divisb n o

fToxbobgba l Researc h an d Testin g NIEH S Researc h Triangl e Park , Nort h Carolin a Elizabet h Grossma n Offic e o fRis k Assessmen t Healt h Standard s Program s U S Departmen t o

fLabor/OSH A Washington , D.C .

## Externa l Pee rReviewers (cont. ) Externa l Pee rReviewer s (cont. )

Gordo n Hard (Criteri a Workgrou p Chair) 8 Medica l Researc h Counci l ToxicologyUni t Carshalton , Surrey Great Britai n Loi s Lehman-McKeeman 8 Th e Procte ran d Gambl e Compan y Miam i ValleyLaboratorie s Cincinnati , Oh b Denni s Lync h Natbna l Institute fo r Occupatbna l Safetyan d Healt h Divisio n o

fBbbgica l an d Behavbra l Science s Cincinnati . Oh b James McKinne

y USEP A Healt h Effects Researc h Laborator y Researc h Triangl e Park . Nort h Carolin a Josep h McLaughli n Natbna l Cance rInstitut e Bethesda , Marylan d Michae l Olso n (Nephropath yan d Biochemistr

yWorkgrou p Chair) 8 Genera l Motors Researc h Laboratorie s Warren , Michiga n Norber t P . Pag e (Ris k Characterizatio n Workgrou p Chair) Pag e Associates Gaithersburg , Marylan d Jame s Poop 8 Departmen t ofExperimenta l Patholog yan d Toxicolog y Chemica l Industrie s institute fo r Toxicolog y(CUT ) Researc h Triangl e Park , Nort h Carolin a Car l Potte r USEP A Ris k Reductio n Engineerin g Laborator

y Cincinnati , Oh b Jame s Swenberg 8 Universit yo fNort h Carolin a Departmen t o fPatholog y Chape l Hill . Nort h Carolin a Benjami n Trum p Universit yo fMarylan d Schoo l o l Medicin e Baltimore , Marylan d Jerrok fWar d Natbna l Cance rInstitut e Fredric k Cance rResearc h Cente r Fredrick , Marylan d Laure n Zeis s Californi a Departmen t o l Healt h Service s Reproductive an d Cance r Hazar d Assessmen t Sectb n Berkeley . Californi a O n Marc h 27 . 1991 . EPA' s Environmenta l Healt h Committe e ofth e Scienc e Advisor yBoar d reviewe d an d unanimousl yapprove d a draf t repor t (EPA/625/3-91 / 019A) . Th e FIFR A Scienc e Advisor yPane l wa s represente d b yDr . Jo e H . Grisham . Th e Executive Committe e approve d th e repor t o fth e Environmenta l Healt h Commit te e at thei rJur

y 23 . 199 1 meeting , althoug h on e membe ro fth e Executiv e Committe e encourage d greate rattentio n t o th e uncertaintie s concernin g th e hypothesis . I n summary , th e Scienc e Advisor yBoar d concurre d wit h th e positio n o fth e Ris k Assessmen t Foru m report . Th e Technica l Pane l an d th e Ris k Assessmen t Foru m als o acknowledg e wit h appreciatio n th e specia l contribution s o fJosep h McLaughlin , wh o greatl yassiste d i n th e preparatb n ofth e epidemiolog ysectbn . an d JerryBlancato , Margare t M.L . Chu . Il a Cote , Richar d N . Hill , an d th e member s o fth e Ris k

Assessmen t Forum' s Cance rOversigh t Grou p (Willia m Farland . Edwar d Ohanian . Vaness a Vu , an d Jeanett e Wiltse ) fo rthei rthoughtfu l comments .

* Also reviewed a

preliminary

draft. Other

reviewers

ot this draft were Carl Alden of Proctor and Gamble's

ktami Vatey Laboratories, and

Michael Lipsky of the University

of Maryland

Medical School.

X I

## Prefac e

Th e U.S . Environmenta l Protectb n Agenc y(EPA ) Ris k Assessmen t Foru m wa s establishe d t o promote scientifi c consensu s o n ris k assessmen t issue s an d t o ensur e tha t thi s consensu s i s incorporate d int o appropriat e risk assessmen t guid ance . T o accomplis h this , th e Ris k Assessmen t Foru m assemble s expert s fro m throughou t th e EP A i n a forma l proces s t o stud yan d repor t o n thes e issue s fro m a n Agency-wid e perspective . Fo rmajo rris k

assessmen t activities , th e Ris k Assessmen t Foru m ha s estab lishe d Technica l Panel s t o conduct scientifi c revie wan d analysis . Member s ar e chose n t o assure that necessar ytechnica l expertis e i s available . Outsid e expert s ma yb e invite d t o participat e a s consultant s or , i fappropriate , a s Technica l Pane l members . Th e us e o

fmal e ra t kidneytumor s i n risk assessmen t ha s bee n th e subjec t o f muc h recen t discussion . Fo ra certai n grou p o fchemicals , investigators hav e reporte d rena l tubul e tumo rformatb n i n mal e rat s a s th e sequel a o frena l toxicit

y commencin g wit h a n excessive accumulatb n o fth e protein , alphas-globuli n (o^-g) , i n rena l tubules . Rena l tubul e tumo rformatb n wit h protei n accumulatb n ha s not bee n observe d i n femal e rat s o rothe rteste d species , mos t notabl yth e mouse . Th e NC I Blac k

Reite rrat , whic h doe s no t produc e a^-g , als o fail s t o sho wa proliferative respons e i n th e kidne yo revidenc e o

fa promotiona l effect whe n expose d t o chemical s that induc e protei n droplet accumulatb n i n mal e rat s o fothe rstrains ; it s respons e ha s no

t bee n teste d i n a conventiona l 2-yea ranima l bbassay . Som e scientist s appl yth e observatbn s see n i n animal s t o conclud e tha t an yrena l tubul e tumo ri n mal e rats observe d i n connectb n wit h a^- g accumulatb n i s a species specifi c effec t inapplicabl e t o huma n risk assessment . Othe rscientists argu e tha t mor e informatio n o n human s i s neede d an d tha t al l mal e ra t kidne ytumor s shoul d continu e t o b e considere d a s relevan t t o huma n ris k a s othe rtumors . Becaus e th e questb n i s relevan t i n assessin g risk fo ra numbe ro

fchemical s of interest t o EPA , th e Ris k Assessmen t Foru m establishe d a Technica l Pane l t o assembl e an d evaluat e th e curren t evidenc e an d t o deveb p scienc e policyrecom mendatbn s fo rAgency-wid e use . Thi s documen t i s th e produc t o fthat effort . Th e literature reviewsupportin g thi s documen t i s curren t a s o fJul y1 , 1991 . 

## I . Executive Summar y

Thi s repor

t o fa Technica l Pane l o

fth e U.S . Environmenta l Protectio n Agency(EPA ) Ris k

Assessmen t Foru m describe s condition s unde rwhic h th e Foru m advise s EP A ris k assessor s agains t usin g informatio n o n certai n rena l tubul e tumor s o rnephrotoxicit yt o asses s huma n risk . Ris k assess men t approache s generall yassum e that chemical s producin g tumor s i n laborator yanimal s are a potentia l cance rhazar d t o humans . Fo rmost chemicals , includin g man yroden t kidneycarcinogens , thi s extrapolatio n remain s appropriate . Th e scientifi c studie s reviewe d by th e Technica l Pane l indicate , however , tha t som e othe rchemical s induc e accumulatio n o f alphas-globuli n (a^-g) , a low-molecular-weigh t protein , i n th e mal e rat kidney . Th e OL- g accumulatio n initiate s a sequenc e o fevents tha t appear s t o lea d t o rena l tubul e tumo rformation . Femal e rat s an d othe rlaboratory mammal s administere d th e sam e chemical s d o no t accumulat e low-mo lecular-weigh t protei n i n th e kidneyan d the yd o not develo p rena l tubul e tumors . Sinc e human s appea rt o b e more lik e othe rlaboratoryanimal s tha n lik e th e mal e rat , i n thi s specia l situation , th e mal e rat i s no t a goo d mode l fo rassessin g huma n risk. Th e analysi s o

fth e scientifi c studie s an d relate d scienc e polic yset ou t i n thi s repor

t eac h stres s th e nee d fo rful l scrutin yo fa substantia l set o

fdat a t o determin e whe n it i s reasonabl e to presum e that rena l tumor s i n mal e rat s resul t fro m a proces s involvin g a^- g accumulatio n an d t o selec t appropriat e procedure s fo restimatin g risks to human s unde rsuc h circumstances . Th e repor t als o define s situation s that suggest differen t approache s an d i t call s fo rresearc h t o clarif

yunanswere d question s regardin g th e mechanism s accountin g fo rth e respons e i n mal e rats . Alpha^-globull n an d Rena l Lesion s I n th e mal e rat , th e productio n o frena l tumor s b

ychemical s inducin g alphas-globuli n accumulatio n (CIGA ) i s precede d b yth e rena l lesion s ascribe d t o a^-associate d nephropathy . Th e involvemen t o fhyalin e drople t accumulatio n i n th e earl ynephrotoxicit yassociate d wit h CIG A i s a majo rdifferenc e fro m th e sequenc e see n (o rclassica l carcinogens . Th e pathologi c change s that preced e th e proliferative sequenc e fo rclassica l rena l carcinogen s als o includ e a for m o fearl ynephroxicity , bu t n o apparen t hyalin e drople

t accumulation . Investigation s performe d i n multipl e laboratorie s ove rth e last decad e have demonstrate d a consisten t associatio n betwee n hyalin e droplet s containin g a.- g an d productio n o fcertai n lesion s i n th e mal e rat kidney . Thes e rena l lesion s ar e no t foun d i n mice , femal e rats , o rothe rlaborator y

### specie s tested

. Th e histopathologica l sequenc e i n th e mal e ra t consist s o

f th e following : a n excessiv e accumulatio n ofhyalin e droplet s containin g OL- g i n rena l proxima l tubules ; subsequen t cytotoxicityan d single-cel l necrosi s o fth e tubul e epithelium ; •sustaine d regenerativ e tubul e cel l proliferation , providin g exposure continues ; •developmen t o fintralumina l granula rcast s fro m sloughe d cel l debri s associate d wit h tubul e dilation , an d papillar y mineralization ; •foc i oftubul e hyperplasi a i n th e convolute d proxima l tubules ; an d finally , •rena l tubul e tumors

. Biochemica l studie s wit h mode l compound s showtha t CIG Ao rthei r metabolite s bin d specifically , bu t reversibly , t o mal e rat a^-g

. Th e resultin g a^-g-CIG Acomplexappears t o b e mor e resistan t t o hydrolyti cdegradatio n b ylysosoma l enzyme s tha n native , unboun d ot^-g

. Inhibitio n o fth e catabolis m o

fo^-g , a protei n onl yslowl yhydroryze d b yrena l lysosoma l enzymes unde rnorma l physiologica l conditions , provide s a plausibl e basi s fo rth e initia l stag e ofprotei n overloa d i n th e nephropath ysequence . Compariso n wit h Classica l Carcinogen s I t i s instructiv e t o compar e CIG Arena l carcinogen s wit h othe rrena l carcinogens

. Severa l genotoxi cchemical s recognize d a s classica l inducers o froden t kidneytumors hav e bee n use d t o stud yth e pathogenesi s ofrena l tubul e cance ri n laborator yanimals

. I n general , thes e prolotypi crena l carcinogen s produc e tumors i n bot h male s an d females

. Althoug h th e wid e rang e o fchemical s represente d suggests multipl e mechanism s ofaction , man yo fth e classica l rena l carcinogen s o rthei ractiv e metabolite s are electrophili cspecie s abl e t o bin d covalentl yt o macromolecule s an d likel yt o for m DN Aadducts i n th e kidney

. I n contrast , CIG Arena l carcinogen s are no t know n t o reac

t wit h DN Aan d are generall ynegative i n short-ter m test s fo rgenotoxicity

. CIG Arena l carcinogen s als o interac t wit h a^- g i n a reversibl e an d noncovalen t manner . CIGAproduce d minima l change s i n urin e chemistr yan d ver ylittl e o rn o glomerula rdysfunctio n i n mal e rats

. Th e mil d tubul e toxicityo fCIGA , i n contras t to th e obviou s urinar

ychange s induce d byrena l toxin s suc h a s mercuri cchlorid e o rhexachlorobutadiene , i s characteristi co fCIG Aan d i s consisten t wit h th e notio n that CIG Ad o no t bin d covalentl yt o a^- g Classica l rena l carcinogens , suc h as certai n nitrosamines , induc e rena l tubul e cance ri n rats o rmice wit h hig h incidence , minima l duratio n of exposure , an d clea rdose-respons e relationships

. There i s usuall yn o absolut e sex-specificity , althoug h male s an d female s ma yb e susceptibl e t o differen t degrees

. I n contrast , th e rena l tumor s produce d byth e eigh t mode l carcinogen s examine d i n thi s repor t tende d not t o b e life-threatening , occurre d lat e i n life , usuall ybein g foun d at termina l sacrifice , an d wer e frequentl ymicroscopic

. Eve n thoug h th e maximu m tolerate d dos e wa s exceede d fo rsom e o

fth e eigh t mode l carcinogens , th e rena l tumo r incidenc e rate , adjuste d fo rintercurren t mortality , wa s nevergreaterthan2 8 percent

. A n increas e i n rena l tubul e tumor s wa s not foun d i n mic e o rfemal e rat s expose d t o thes e chemicals

. Initiation/promotio n studie s wit h gasoline , trimethytpentan e (TMP ) an d d-limonen e i n Fische r34 4 rat s showe d tha t thes e CIG Apromote d atypica l tubul e cel l hyperplasi a and/o rrena l tubul e tumors i n male s bu t not i n females

. I n contrast , d-limonen e di d no t promot e thes e lesion s i n male s o fth e NC I Black-Reite r(NBR ) strai n i n th e sam e initiation/promotio n model

. Suc h difference s i n potencyan d species- , strain - an d sex-susceptibilitysuggest (ha t CIG Arena l carcinogen s ac t vi a differen t mechanism s tha n classica l rena l carcinogens

. Rena l tubul e tumors produce d byCIG Acarcinogen s als o hav e feature s i n commo n wit h othe rrena l tubul e tumors observe d i n th e mal e rat

. Fo rrena l carcinogens , i n general , there i s a continuu m ofchemicall yinduce d step s fro m atypica l hyperplasi a throug h microscopi cadenoma s t o macroscopi c adenocarcinoma s o rcarcinomas

. Rena l tubul e tumors induce d b yth e eigh t mode l carcinogen s are morphologicall yindistinguishabl e fro m thos e in duce d b yclassica l carcinogens

. Likewise , th e sequenc e ofdevelopmen t o f CIG Acarcinogen-induce d rena l tumors fro m tubul e cel l hyperplasi a t o carcinom a appears identical

. Furthermore , non e o fthes e chemicall y induce d tumors ca n b e differentiate d fro m spontaneou s tumors

. Othe rConsideration s Al l eigh t o

fth e mode l carcinogen s examine d i n thi s repor t wer e als o capabl e ofproducin g rena l tubul e hyperplasi a i n mal e rats

. I n general , thi s hyperplasi a becam e mor e severe wit h increasin g dose

. Th e occurrenc e of thes e preneoplasti clesion s togethe rwit h th e neoplasti clesion s provide s indirect evidenc e ofprogressio n tha t i s i n accor d wit h generall yaccepte d view s o n rena l tubul e tumo rformatbn

. Dose - an d time-relate d association s betwee n th e administratio n o f CIG At o mal e rat s an d th e variou s histologica l stage s hav e bee n observed

. Thes e relationship s were demonstrate d betwee n CIG Aadministratio n fo r bot h hyalin e drople t formatio n an d a, u 

### - g accumulation

. Althoug h th e relationship s betwee n increase d hyalin e droplet s an d cel l necrosi s o r betwee n cel l necrosi s an d cel l regeneratio n hav e not bee n quantified , a correlatio n betwee n hyalin e drople t respons e an d th e numbe rofcell s excrete d i n th e urin e has bee n observe d fo rCIGA

. Dose-respons e relation ship s betwee n hyalin e droplet accumulatio n an d proxima l tubul e cel l prolif eratio n have bee n show n fo rTM P an d unleade d gasoline

. Clea rdose respons e relationship s wer e demonstrate d betwee n linea rmineralizatio n i n th e rena l medull a an d incidenc e o frena l tubul e neoplasi a i n mal e rat s i n severa l bioassays

. Arecen t stud yo i d-limonen e demonstrate d a relation shi p betwee n severit

yo fnephropath yan d rena l tubul e cance ri n mal e rats

. Th e Technica l Pane l i s no t awar e ofan yepidemiologi cstud ythat ha s bee n designe d o rconducte d specificall yt o examin e th e applicabilityo fth eCIGA hypothesi s t o rena l cel l cance ri n humans . Severa l epkJemblogi c studie s wer e reviewe d fo rthi s report , but theyar e o flimite d valu e fo rthi s analysi s becaus e the yinvolve d exposure t o complexblends , suc h a s gasoline , o rotherwis e involve d multipl e exposure s t o bot h CIG A an d non CIGA . I n addition , thes e studie s wer e o flimite d statistica l powe ran d wer e no t abl e t o accoun t fo rpossibl yconfoundin g factors , suc h a s smokin g o r obesity , whic h ar e know n to influence rena l cel l cance rrates . I n a fe w studies , sligh t increase s i n ris k o frena l cel l cance rhav e bee n observed ; however , th e othe rfactor s describe d above coul d hav e easil yaccounte d fo r (h e increase d risk. Thes e studies , therefore , ar e considere d inadequat e fo r purpose s o

fexplorin g th e relevanc e o fth e a^- g hypothesi s i n humans . Low-molecular-weigh t protein s that probabl yhav e a three-dimensiona l structur e simila rt o a^- g have bee n identifie d i n mic e an d othe rspecies , includin g humans . I n vitro studie s hav e show n tha t th e active metabolit e o f TM P form s complexe s wit h som e o fthes e proteins . Othe ri n vitr o studie s indicate , however , tha t reversibl e bindin g doe s no t necessaril yincreas e resistanc e t o hydrolyti c degradation , a featur e apparentl yrequire d fo r hyalin e droplet formation . Extensive studie s o

fmice , whos e urin e contain s larg e amount s o f mous e majo rurinar

yprotein s (MUP) . hav e foun d n o evidenc e o frena l lesion s simila rt o thos e associate d wit h th e a^- g syndrome . Thus , th e presenc e o

fa structurall yrelate d protein , eve n i n larg e quantitie s i n th e urine , doe s no

t impl ythat anothe rspecie s wil l respon d i n a manne rsimila r to th e mal e rat . Th e lor m o

fO L - g that originate s i n th e live ro fth e mal e rat i s no

t detecte d i n th e femal e rat . Tik e th e mouse , th e femal e rat shows n o evidenc e o fa n a^-g-like nephropath ywhe n expose d to CIGA . I n case s wher e nephrotoxicit y wa s observe d i n mic e o rfemal e rats , i t wa s les s sever e o rqualitativel y differen t fro m that i n mal e rat s an d di d no t involv e th e spectru m o

fdiscrete lesion s associate d wit h a^- g accumulatio n i n th e mal e rat . Specialize d studie s o

frats , suc h a s thos e involvin g immature , age d an d castrate d mal e rats , male s o fth e NC I Blac k

Reite r(NBR ) strai n (whic h doe s no t synthesiz e a^- g i n th e liver) , an d injectio n ofmal e rat s wit h estroge n an d femal e rats wit h a -g , sho wtha t developmen t o

fth e earl yfeature s o

fth e specifi c nephropath ysyndrom e occurs onl yi n th e presenc e o fO L - g . Very limite d informatio n fro m dogs , hamsters , guine a pigs , an d monkey s als o supports thi s statement . Thes e studie s furthe rsuppor t th e hypothesi s that thi s o^-g-relate d nephropath yoccurs specificall yi n th e mal e rat . Summar y I n summation , th e reversibl e bindin g o fth e compoun d t o O L -g , whic h result s i n a shif

t i n balanc e betwee n reabsorptio n an d hydrolysi s an d th e accumulatio n o

fa - g i n hyalin e droplets i n th e P 2 segmen t o fth e rena l tubule , provide s a plausibl e explanatio n fo rth e initia l step s i n a sequenc e o f event s leadin g t o th e formatio n o frena l tubul e tumors i n th e mal e rat . A sustaine d protei n overloa d woul d resul t i n single-cel l necrosi s i n th e tubul e epitheliu m an d increase d cel l regeneration , wit h granula rcast form a papillar ymineralizatio n a s indirec t consequences . Th e increase d \. tiv e respons e cause d by chemicall yinduce d cytotoxicit ymayb e a r

. reaso n fo rth e developmen t o frena l tubul e tumo ri n mal e rats . Th i tubul e tumor s produce d i n mal e rat s i n associatio n wit h CIG A o v g nephropath yshoul d b e distinguishe d fro m othe rrena l tubu k i n term s o

fus e i n huma n ris k

assessment .

oII . Introductio n

Fo rmos t hazardou s chemicals , adequat e huma n dat a ar e no t available , an d ris k analyse s mus t rel yo n informatio n fro m laborator ystudie s o frat s o r mice . Th e inferenc e tha t th e result s ofanima l experiment s ca n b e applie d t o human s i s a fundamenta l principl e o fal l toxicologi c research . Thi s pape r deal s wit h a specifi c case , however , wher e th e mal e ra t seem s t o respon d i n a differen t manne rtha n othe rlaborator yspecies . Th e possibilit yo fa uniqu e respons e i n th e ra t amon g laborator yanimal s raise s question s abou t th e applicabilit yo fcertai n ra t dat a t o othe rspecies , includin g humans . Thi s documen t provide s guidanc e fo rth e assessmen t o fsuc h information . A variet yo forgani c chemical s hav e produce d specifi c rena l lesion s i n mal e rat s i n th e for m o fa hyalin e drople t nephropath yaccompanie d b y accumulatio n o fth e protein , alphas-globuli n (OL-g ) (Healt h Effect s Institut e [HEI ] 1985.1988) . Amon g th e chemical s teste d ar e paraffin s (Haide re t al. , 1984 ; Phillip s an d Cockrell , 1984) , decali n (decahydronaphthalene ) (AkJe n e t al. , 1984 ; Kanerv a e t al. , 1987a) . petroleum-base d an d syntheti c fuel s (MacNaughto n an d Uddin , 1984) . militar yaviatio n propellant s (Bruner , 1984 ) an d 2,2,4-trimethylpentan e (TMP ) (Haide re t al. , 1985) . A s see n i n Tabl e 1 . whic h list s a samplin g ofchemical s tha t hav e bee n tested , man yar e ofconsiderableregulatoryandcommercialinterest . Fo rexample , isophoron e i s a chemica l intermediat e o fmajo rindustria l importance . Aviatio n an d automotiv e fuel s fi t int o th e category , a s doe s th e natura l foo d product , d-limonene , foun d i n citru s oils . Thi s analysi s focuse s o n mode l compound s havin g bot h a n adequat e anima l carcinogenesi s bioassa yan d informatio n o n OL^- g o rhyalin e drople t accumulatio n i n th e mal e rat . Thes e substance s ar e seve n chemicals , 1,4 - dichlorobenzen e (1,4-DCB) , dimethy l methy l phosphonate , hexachloroeth ane , isophorone , d-limonene , pentachloroethane , an d tetrachloroethylen e an d a mixture , unleade d gasoline . Thes e eigh t substance s ar e compare d an d contraste d wit h tw o relate d no n cts-g-inducers , chlorothaloni l an d Irichloroethylene . Th e analysi s als o relie s o n researc h studie s o n tw o othe r mode l compounds , decali n an d TMP , whic h hav e extensiv e informatio n o n a^- g nephropath ybu t n o chroni c bioassa ydata . Mor e limite d informatio n o n 2 4 additiona l substance s i s als o discusse d wher e appropriate . O fth e eigh t mode l substance s teste d i n chroni c anima l bioassays . al l invoke d a specifi c typ e o fprotei n drople t nephropath yi n mal e rat s an d als o produce d rena l tumor s i n mal e rat s bu t no t i n othe rspecie s tested . I t ha s bee n propose d tha t suc h rena l tumor s ar e th e en d produc t i n th e followin g sequenc e offunctiona l change s i n th e epithelia l cell s o fproxima l tubule s (Universitie s Associate d fo rResearc h an d Educatio n i n Patholog y(UAREP] , 1983 ; Alde n e t al. , 1984 ; Haide re t al. , 1984 ; HEI , 1988 ; Swenber g e t al. , 1989) . Excessiv e accumulatio n ofhyalin e droplet s i n proxima l tubules , representin g lysosoma l overload , lead s t o tubul e cel l degeneration , cel l loss , an d regenerativ e cellula rproliferation . Tmbim 1 Examples

ol Organic

Chemicals

that have Produced

Renal Injury in Mate > r^cTancterixedby Hyaline

Droplet Accumulation

but not m Female Rats or other Species. Chemica l Specie s Teste d Decali n Rat s Mic e Dog s Guine a pig s (m/l )

<m/f ) 

### M ) <m/l ) Rena l

Toxicit y +/ - -/ - -/ - Referenc e

Alde n e l a l (1965 ) USEP A (1967 )

Dimetfiy l methyl phosprmnal e Isophoron e JP- 4 je t fue l

JP- S shale-derive d je t fue l d-IJnwnen e Methy l isobuty l keton e

Pentachloroethan e Unleade d gasolin e

m -mal e f-femal e

•- positive

- -negativ e Rat s

Mic e Rat s Mic e Rat s Mic e Dog s Rat s Mic e Dog s Ra U Mic e Dog s Rat s Mic e Dog s Monkeys Rat s Mic e Rat s Mic e (nvT

) (m/l ) (m/l ) (m/l ) <m/l ) (m/l ) (m/l ) (m/f ) <m/ 0 (m/f ) (m/t ) (m/l ) (m/f ) (m/t ) (m/f ) (m ) (m ) (m/f ) (m/l ) (m/f ) (m/l ) • / -

-/ - */- -/ - W - -/ - -/ - •/ - -/ - -/ - +/ - -/ - -/ - •/ - -/ -

•/ - -/ - W - -/ - NT P (1987b )

NT P (1986a ) MacNaughto n an d Uddi n (1984 ) MacNaughto n an d Uddi n (1984 ) NT P (1990 ) Web b e t at (1990 ) Alde n etal . (1984 ) Phillip s e t al . (1987 ) NT P (1983 ) USEP A (1987 )Cel l debri s i n th e for m o fgranula rcasts accumulate s at th e "corticomedullary " junctio n wit h associated dilatio n o fth e affecte d tubul e segmen t an d more distally , mineralizatio n o ftubule s withi n th e rena l medulla . Single-cel l necrosi s accompanie d b ycompensatorycel l proliferatio n an d exacerbatio n o fth e chroni c progressiv e nephropath y(CPN ) characteristicall yfoun d i n agin g rat s occurs . Rena l tubul e hyperplasi a an d neoplasi a develo p subsequently . Accordin g t o thi s hypothesis , th e increase d proliferative respons e cause d b

y th e chemicall yinduce d cytotoxicit yresults i n clona l expansio n o f spontaneousl yinitiate d rena l tubul e cell s an d increase d incidenc e o frena l tumo rformatio n (Trum p e t al. , 1984a ; Alden , 1989 ; Swenber g et al. , 1989) . Thi s lin e o

freasonin g lead s supporters o fth e hypothesi s t o conclud e tha t th e acute an d chroni c rena l effect s induce d i n mal e rat s b ysuc h chemical s wil l b e unlikel yt o occu ri n an yspecie s no t producin g OL^-g , o ra veryclosel y relate d protein , i n th e larg e quantitie s typicall ysee n i n th e mal e ra t (Alde n 1989 ; Borghof

fetal. . 1990 ; Gree n etal. , 1990;Olso n etal. . 1990 ; Flam m an d Lehman-McKeeman , 1991 ; Swenberg , 1991) . Thi s repor

t examine s th e hypothesi s tha t th e mal e rat i s predispose d t o th e nephrotoxi c effect s induce d bycertai n classe s o fchemicals , suc h a s volatil e light hydrocarbon s an d organohalkJes . It als o examine s dat a tha t suppor t o rcontradict th e concep t that th e rena l tumors produce d i n mal e rat s b y thes e chemical s ar e causall yrelate d t o th e nephrotoxicity . Base d o n th e Ris k Assessmen t Forum' s (RAF ) conclusbn s regardin g thes e data , th e documen t describe s a unifor m approac h fo rEPA t o us e i n ris k assessment s dealin g wit h thi s spectru m o flesion s an d categor

yo fchemicals . Informatio n fo rthi s RA F repor t wa s obtaine d initiall yfro m a 198 8 revie w entitled , "Evaluatio n o

fDat a Concernin g th e Relationship s amon g Chemi cally-induce d Rena l AlphjLGIobuli n o rHyalin e Drople t Accumulation , Nephropathy , an d Rena l Neoplasia " prepare d fo rth e Offic e ofToxi c Substance s b

yDr . Willia m Richard s o fDynama c Corporation , Rockville , Maryland . Additiona l informatio n considere d i n thi s repor t include s recen t comprehensive reviews o fth e subject , comments fro m pee rreviewers , an d othe rorigina l work , especiall ypublication s subsequen t t o th e 198 8 Dynama c review . Thi s repor

t ha s fou rparts . Followin g thi s brie fintroduction , Part 1 addresse s th e characteristic s o fhyalin e droplets , th e protein , ct^-g , an d th e nephropath yassociate d wit h a^ g accumulatio n (Section s II I an d IV) . Par t 2 (Section s V-VIII ) present s dat a o n th e carcinogeni c potentia l o f chemical s inducin g alphas-globuli n accumulatb n (CIGA ) i n th e mal e rat . Sectio n V describe s th e preneoplasti c an d neoplasti c lesion s produce d by classica l rena l carcinogens . Sectio n V I considers generi c factor s relevan t t o al l studies o

fpotentia l rena l carcinogenicit yi n laborator

yanimal s an d the n analyze s an d discusse s dat a o n th e rena l lesion s observe d i n 2-yea r bioassays wit h chemical s causin g th e hyalin e drople t nephropathy . Sectio n VI I examine s additiona l informatio n tha t assists i n definin g rena l carcino gen s a s CIGA , i n particula rgenotoxicil y an d initiation-promotio n data . I n Sectio n VIII , CIG A ar e compare d wit h classica l rena l carcinogens , whil e Sectio n I Xconsider s th e huma n evidenc e fo rkidney cancer , its histogenesi s an d epidemiology . Sectb n Xexamine s evidenc e fo rth e hypothesize d dose - an d time-dependen t progressb n o flesbns . Par t 3 evaluate s th e evidenc e considere d i n Part s 1 an d 2 wit h regar d t o th e hypothesi s tha t OL- g accumulatio n i n th e kidneyi s a n initia l ste p i n a successio n o

fhistopathologi c event s that mayculminat e i n rena l tubul e tumo rformatio n i n mal e rals . Thi s par t als o lists prioritie s fo rfuture research . Par t 4 comprise s th e Agencypolic ystatemen t regardin g approache s t o ris k assessmen t fo rthi s categor yo fchemicals . Fo rclaritythroughou t th e review , nomenclature i s standardized , an d abbreviation s are use d fo rfrequentl yrepeate d terms . Insofa ra s hyalin e droplet represent s a morphologica l entit yrequirin g onl yligh t microscopyfo r identification , thi s ter m wil l b e use d i n preferenc e to th e synonymou s protei n droplet' . Th e designatbn , o^- g nephropath yi s use d t o connot e th e ful l sequenc e o

fpathologi c lesion s fro m hyalin e droplet formatio n t o restorative hyperplasi a an d medullar ymineralizatbn . Toxi c tubula rnephropath yi s a nonspecifi c ter m commonl yuse d i n roden t bioassa yreport s t o describ e variou s form s o

fnephrotoxicit

yinduce d b

ychemicals , includin g th e specifi c lesion s o fa^- g nephropathy . Th e spontaneou s age-relate d syndrom e o f ra t kidneydiseas e otherwis e know n i n th e literatur e a s ol d rat nephropathy , chroni c nephrosis , glomerulosclerosis , an d progressive glomerulonephrosis , i s standardize d accordin g t o Barthol d (1979 ) a s chroni c progressiv e nephropath y(CPN) . Th e ter m lipocali n i s use d accordin g t o th e terminolog y o fPervai z an d Brew(1987 ) t o describ e th e superfamil yo ftow-molecularweigh t protein s whic h appea rt o transpor t lipophili c substances . I n rats , th e proxima l tubul e o fth e nephro n i s divisibl e morphologicall y into thre e part s (se e Figure 1) . Th e first segmen t i s i n continuitywit h th e parieta l epitheliu m o l Bowman' s capsul e surroundin g th e glomerula rtuft . Together , th e first an d secon d segment s represen t th e convolute d portio n o fth e proxima l tubul e an d ar e situate d wholl yi n th e cortex , th e outermos t zon e o fth e rat kidney . Th e thir d segmen t i s th e straigh t portio n o fth e proxima l tubul e (par s recta ) comprisin g th e oute rstrip e o fth e oute rmedull a bu t als o th e medullaryrays arisin g i n th e cortex . Th e abbreviation s P1 , P2 , an d P 3 are use d conventionall yt o denot e thes e thre e segments . Th e ter m rena l tubul e tumo rdescribe s neoplasm s o fth e rena l cortica l tubul e epithe liu m comprisin g collectivel yadenoma , adenocarcinoma , an d carcinom a accordin g t o standardize d nomenclature determine d byth e Societyo f

Hyalin e droplet s rele rt o spherica l inclusion s i n th e cytoplas m whic h ar e homogeneou s an d eosinophilic , representin g overdstende d phagolysosomes . The y ma ycontai n variou s macromolecuie s includin g o^-globulin . Th e morpholog yo l droplet s containin g differen t protein s me , o e identica l an d therefore immunocytochemistr y i s require d to r precis e definitiio n o

t contents .Thic k ascendin g ImbofHenl e

Thi n descendin g lim b o fHenl e

Thi n ascendin g lim b o l Henl e

Figure 1. Diagram

of Zonation

and Tubule Segmentation

in Rat Kidney. G:

Gtomerutus: PI: First segment

of proximal convoluted

tubule; P2: Second segment

of proximal convoluted

tubule: P3: Pars recta of proximal tubule. Source: Adapted from Bachmann

et al. {1986). 

### Toxicologi cPathologists (Hard et al. , 1991)

. Excep t whe n specified , th e term s adenocarcinom a an d carcinom a ar e use d interchangeably

. Th e sam e neoplasm s are referre d t o as rena l cel l tumors i n humans , i n keepin g wit h th e genera l literature (Bannaya m an d Lamm , 1980)

. 1 0 

## Parti . Nephrotoxicit y

III . Hyalin e Droplet s an d Alpha ^ - Globulin ; Physiolog yan d Biochemistr

y Informatio n o n th e rena l processin g oflow-molecular-weigh t proteins , sexan d specie s difference s i n urinar yproteins , an d th e characteristic s o f a^- g provide s a n explanator ybasi s fo rth e accumulatio n ofot^- g i n hyalin e droplet s i n th e mal e rat followin g exposur e t o CIGA

. I t i s pertinent , therefore , t o examin e th e physiologica l an d biochemica l characteristic s o fa^- g an d relate d proteins , particularl ythos e tha t occu ri n humans , befor e explorin g th e possibl e association s betwee n a^- g accumulation , rena l toxicit yan d rena l tumo rformatio n an d thei rrelevanc e t o huma n ris k assessment

. A. Filtration, Reabsorption, and Catabolism

ofLow-MolecularWelght Proteins

by tha Kidney Th e mammalia n kidneyha s a majo rrol e i n maintainin g th e plasm a concentration s ofcirculatin g low-molecular-weigh t protein s a t thei rnormall y tow, physiologica l level s Thus , low-molecular-weigh t protein s are continu all yremove d fro m th e plasm a b yglomerula rfiltratio n followe d b yreabsorptio n an d catabolis m i n th e proxima l tubule s (Maac k et al. , 1985 ) o rb yexcretion

. Figure 2 i s a schemati crepresentatio n o fth e cellula ruptake an d dispositio n o ffiltere d protein s b yth e rena l tubule

. Th e norma l rena l gtomerulu s freel ypasse s protein s wit h a molecula r weigh t o fles s tha n 20,00 0 dattons , includin g peptide s suc h a s insulin , rysozyme , rat growt h hormone , myoglobin , an d cytochrom e C (Maac k e t al. , 1985)

. Fo rlarge rprotein s lik e th e albumin s an d globulins , whic h hav e a fa r greate rplasm a concentratb n an d muc h lowe rfiltratio n rat e tha n low molecular-weigh t proteins , th e kidne yha s n o regulatin g rol e i n plasm a protei n concentratbn

. Reabsorptio n o

ffiltere d protei n occurs predominantl yi n th e convolute d par t ofth e proxima l tubul e an d t o a lesse rexten t i n th e par s rect a cells

. Tubula rabsorptio n o fa protei n i s a comple xproces s initiate d b ybindin g o f th e protei n t o th e microvill i o fth e proxima l tubul e epithelium

. Thi s i s followe d bymigratio n t o th e bas e ofth e microvill i an d adsorptiv e endocytosi s wherebyinvaginatio n o

fth e surfac e membran e internalize s th e protei n (Kayse n et al. , 1986)

. Whil e reabsorptio n wa s onc e considere d largel y nonselective , hig h capacity , lowaffinitytranspor t (Maac k et al. , 1985) , fro m recen t wor k

i t no wappear s tha t interactio n betwee n th e protei n an d th e brus h borde rmembran e i s th e ste p a t whic h a degre e o

fselectivityi n th e absorpuo n proces s occur s (Kayse n et al. , 1986)

.

1 1

### Tubul e Lume n

Bloo d \ Proxima l (P2 ) Tubul e Cel l

Figure 2. Schematic

representation

of endocytic

uptake of filtered proteins. Filtered proteins are adsorbed

to endocytic sites

at the lumenal membrane

and segregated

in endocytic

vacuoles

(EV). These

EV migrate to the eel interior, where they fuse with tysosomes (L)

to form secondary

lysosomes

(SL) or phagolysosomes

where digestion

of the protein takes place. The

products

of hydrolysis

(amino acids) permeate

the SLmembrane, cross

the contralumenal

ceK membrane, and

return to the circulation. SOURCE:

Adapted

from Kaysen et al. (1986). Withi n proxima l tubul e cells , endocyti c vesicle s fus e t o for m endocyti c vacuole s whic h i n tur n coalesc e wit h lysosome s derive d fro m th e Gokj i apparatus , formin g secondar ylysosomes . Th e hydrolysi s o fprotein s by proteas e enzyme s take s plac e withi n th e secondarylysosomes . Th e lysosoma l enzyme s o

frena l cortica l tubule s includ e two majo rclasse s o f aci d proteinases , i.e. , cystein e proteinase s (cathepsi n B , H , an d L ) an d a n asparti c aci d proteinase , cathepsi n D (Lehman-McKeema n e t al. , 1990a) . Lehman-McKeema n e t al . (1990a ) hav e show n that bot h o fthes e endopep tidas e classe s contribute to th e degradatio n o fo^-g . Lysosome s hav e a large , bu t no t unlimited , capacit yt o cop e wit h increase d amount s o fhydrolyzabte proteins , bu t th e protein s diffe rI n susceptibilityt o hydrolysis . Protei n half-lives , whic h are indice s o fthei r catabolis m byprotease s i n th e kidney , depen d o n specifi c molecula r determinant s i n th e protein . Th e primar yamin o aci d sequenc e ma yb e on e importan t facto ri n determinin g protei n half-live s (Dice , 1987) . Th e plasm a half-live s o

fman ylow-molecular-weigh t protein s are measure d typicall yi n minute s (Maac k

et al. , 1985) . On e o

fth e exception s i s ot^-g , wit h a half-life measure d i n hours (Geertze n e t al. , 1973) . Whethe ro rno

t low-molecular-weigh t protein s lik e ot^- g accumulat e i n kidne ytubule s depend s o n th e balanc e betwee n th e rat e o freabsorptio n by epitheliu m an d th e rat e o fhydrolysi s i n th e cells . Base d o n th e informatio n presente d below , i

t i s believe d that exposur e t o CIG A result s i n a shif t o fthi s balanc e i n mal e rals . B. Hyaline

Droplets

In Renal Tubules Th e product o

fprotei n reabsorptio n an d accumulatio n i n rena l tubul e cell s i s visualize d b

ylight microscopya s hyalin e droplets . Smal l protei n reabsorptio n droplet s o funifor m siz e ar e a constitutive featur e o fnorma l matur e mal e rat s bein g particularl yeviden t i n th e P 2 segmen t o fproxima l tubule s (Logothetopoulo s an d Weinbren , 1955 ; Maunsbach , 1966a ; Goldsworth ye t al. , 1988a) . Ultrastructurally , hyalin e droplet s ar e abnormall ylarge , dense , secondar ylysosome s (als o terme d phagolysosomes) , representin g fusio n o

fendocyti c vacuole s wit h primarylysosomes . Som e hyalin e droplet s sho

wcrystalloi d change s b yelectro n microscopytha t are not observe d i n th e lysosome s o ffemal e rat s (Maunsbach , 1966b) . Crys tallin e formatio n i n th e norma l mal e rat i s believe d t o indicat e th e presenc e o fa poorl ycatabolize d protei n i n pur e solutio n (Peac e e t al. , 1980) , presumabl ya^- g i n th e kidneylysosomes . Hyalin e droplet s I n th e proxima l tubule s o fnorma l mal e rat s contai n cis- g (Alde n e t al. , 1984 ; Gar g et al. , 1987 ; Goldsworth yet al. , 1988a) , an d thei roccurrenc e appear s t o paralle l th e variabl e synthesi s o fthi s protein . Thus , hyalin e droplet s becom e apparen t i n mal e rat s a t th e tim e o fpuberty , bu t the ydeclin e progressivel ywit h increasin g ag e afte r1 8 month s (Logothetopoulo s an d Weinbren , 1955 ; Murty e t al. . .1988) . I n femal e rats , protei n droplet s i n proxima l tubule s ar e eithe rabsen t o rconsiderabl yles s frequen t tha n i n males , an d theyd o no t contai n cL- g (Logothetopoulo s an d Weinbren , 1955 ; Maunsbach . 1966a ; Goldsworth ye t al. , 1988a ; Burnett e t

### al. , 1989)

. Hyalin e droplet s are substantiall yreduce d i n castrate d mal e rat s

(Logothetopoulo s an d Weinbren , 1955)

. Becaus e a n abnorma l increas e i n hyalin e droplet s ha s mor e tha n on e etiolog yan d ca n b e associate d wit h th e accumulatio n o fdifferen t proteins , i t i s necessar

yt o appl yspecia l diagnosti cmethod s suc h as immunohisto chemica l stainin g t o make th e associatio n betwee n chemica l exposure an d pathologi caccumulatio n ofot^-g

. Abnorma l accumulatio n o fhyalin e droplet s i n roden t kidneyi s see n i n certai n diseas e processes . Bot h mal e an d femal e rat s wit h histiocyti c sarcom a showhyalin e drople t accumulatio n i n th e proxima l tubules , indis tinguishabl e fro m th e CIGA-induce d lesion

. Th e accumulatin g protei n i n thes e tumor-bearin g animal s ha s bee n identifie d a s lysozym e (Har d an d Snowden , 1991)

. Similarly , i n mal e an d femal e mice wit h histiocyti ctumors , abnorma l accumulatio n oflysozyme-containin g hyalin e droplet s sometime s occurs i n proxima l tubule s (Har d an d Snowden , 1991)

. I n humans , th e Bence-Jone s proteins , a clas s o fligh t chai n immuno globulins , are produce d i n larg e amount s i n multipl e myelom a patient s (Piran i et al.

. 1983)

. I n huma n case s ofmononuclea rcel l leukemia , lysozym e i s produce d (Muggi a et al. , 1969)

. Th e kidneyinjur ysee n wit h thes e neoplasti cdisease s ha s bee n describe d a s simila rt o that produce d byadministratio n o

fdecali n t o mal e rat s (Alden , 1986) , includin g protei n drople t accumulatio n i n rena l tubule s (Olive ran d MacDowell , 1958 ; Pruzansk i an d Platts , 1970 ; Piran i et al. , 1983)

. Patient s wit h epidemi chemorrhagi c fever , infuse d wit h larg e amount s ofconcentrate d huma n seru m albumi n a s a therapeuti cprocedure fo rshoc k have als o develope d a comparabl e for m o fhyalin e drople t accumulatio n (Olive ran d MacDowell , 1958)

. C. Factors

Affecting

Kidney Accumulation

of Low-Molecular' Weight Proteins Protei n accumulatio n i n th e proxima l tubul e ca n reac h pathologica l level s resultin g i n excessive hyalin e drople t formatio n fo rsevera l reasons : (1 ) th e rat e o

fprotei n deliver

yt o th e tubul e cell s i s abnormall yhigh , (2 ) th e protein s delivere d are difficul t t o hydrolyze , o r(3 ) th e lysosoma l hydrolyz e capacityi s sufficientl yreduced

. Th e rat e o

fprotei n deliver

yt o th e tubul e ca n b e abnormall yhig h unde r condition s whe n th e capillar ywal l o fth e glomerulu s fail s t o provid e th e norma l filtratio n barrier

. Thi s happens , fo rexampl e whe n there i s immuno logical , inflammatory , o rtoxi cdiseas e i n th e glomerulu s o rwhe n th e permselectrvit

ybarrie ri s overloade d b yfilterabl e protein s (Kayse n et al. , 1986)

. Th e increase d urinar

yexcretio n o

flow-molecular-weigh t protein s see n i n disease s suc h a

s multipl e myelom a i n human s o rhistiocyti csarcom a i n rat s i s primaril yth e resul t ofa n increas e i n plasm a concentratio n cause d b y overproductio n o

fspecifi csmal l protein s (Maac k et al. , 1985)

. Lysozym e (histiocyti csarcoma) an d ligh t chai n immunoglobulin s (multipl e myeloma ) ar e protein s that are relativel yresistan t t o hydrolysi s (Maac k e t al , 1985)

. Thi s suggests a combinatio n o frat e o fdeliver yan d difficultyo l hydrolysi s a s etiotogi cfactor

s i n th e accumulatio n o flysozym e i n rat s wit h histiocyti c sarcom a an d ligh t chai n immunoglobulin s i n human s wit h multipl e myeloma

. Th e combinatio n o

fdifficul t hydrolysi s o

fth e protein , a s suggeste d b yit s lon g half-life , couple d wit h hig h rat e o fprotei n deliver yt o tubul e cell s i n th e sexuall ymatur

e mal e ra t als o appears t o b e a facto ri n th e accumulatio n of o^ g i n th e rena l tubule s o fmal e rats

. Th e proces s o

fprotei n hydrolysi s ca n b e reduce d o rinhibite d whe n lysosome s are unabl e t o maintai n th e lowp H require d fo rhydrolyti cenzym e (unction

. Inhibitio n o

fth e metabolicall ydrive n hydroge n io n pump , b y metaboli cpoison s o rth e presenc e o fa wea k bas e i n tubul e lysosomes , alter s th e p H an d result s i n th e accumulatio n o fprotein s (Maac k

e t al.

. 1 985) . I n th e presence ofa reduce d lysosoma l hydrolysi s capacity , th e mos t hydrolyticail yresistan t proteins , lik e o^g , ten d t o accumulat e first

. Testos teron e i s know n t o hav e a suppressiv e effec t o n th e activityo fsom e majo r proteolyti cenzyme s i n th e mal e ra t kidne y(Kugle ran d Vornberger , 1986)

. Consequently , th e lysosoma l proteas e activityi n mal e proxima l tubule s i s lowe rtha n that o

ffemale s (Jedrzejewsk i an d Kugler , 1982 ; Kugle ran d Vornberger , 1986) implyin g tha t th e mal e ra t coul d b e intrinsicall ymor e pron e t o protei n overloa d i n th e rena l tubule s tha n th e femal e rat

. Reductio n o

fth e hydrolyti ccapacityo frena l lysosome s an d increase d resistance o

fprotei n t o hydrolysi s ca n bot h b e affecte d b yexogenou s chemicals

. Althoug h CIG Ama ynot compromis e kidne ylysosoma l enzym e activit ype rse (Murt

ye t al. , 1988 ; Lehman-McKeema n et al. , 1990a) , an y chemicall yinduce d impedimen t t o o^ g digestibilitycause d byCIG Awoul d b e furthe rsuperimpose d o n th e cause s considere d previousl ytha t alon e ca n resul t i n excessive protei n accumulatio n i n rena l tubules

. D. The Alphas-globulin

Superfamily

of Proteins Alphas-globuli n * i s a membe ro fa larg e superfamil yo flow-molecular weigh t proteins

. Th e complet e amin o aci d sequenc e ofou^- g wa s firs t deduce d b

yUnterma n e t al

. (1981)

. Wit h th e exceptio n o fo^- g an d mous e majo rurinar

yprotein(s ) (MUP) , th e sequenc e homolog ybetwee n an ypai r o fprotein s i n thi s superfamil yi s small , abou t 2 0 percen t (Akerstro m an d Logdberg , 1990)

. Statistica l analysi s shows , however , that th e protein s ar e relate d evolutionary(Akerstro m an d Logdberg , 1990)

. O fth e approximatel y2 0 protein s nowconsidere d t o b e potentia l member s o fth e superfamil y(Akerstrd m an d Logdberg , 1990) , th e three dimensiona l structure i s know n fo ronl ythree , retinol-bindin g protein , Blactoglobulin , an d insecticyani n (Sawyer , 1987)

. Th e centra l cor e o fthes e

•Th e mal e ra

t urin e protein , alpha , -globuli n i s name d i n accordanc e wit h immunoelectro phoreti c nomenclatur

e (Ro yan d Neuhaus . 1967) . Becaus e o fits size , som e author s rete rto thi s protei n a s alpha , - microglobuli n o ralph a 2u. - globulin , a ter m mor e appropri atel yreserve d fo ranothe rlow-molecular-weigh t endometria l protei n associate d wit h pregnancy . 1 4 1 5thre e protein s i s compose d o feigh t strand s wit h a B-barre l structur e formin g a hydrophobi c pocke

t tha t appear s t o enclos e th e ligan d (Papi z e t al. , 1986 ; Sawyer , 1987) . Thi s structur e ha s bee n describe d a s resemblin g a coffe e filte rpape r(AkerstrO m an d Logdberg , 1990) . I n additio n t o theb-structura l motif , on e helica l rod an d severa l othe rstructura l element s appea rt o b e conserve d amon g th e proteins . Protei n foldin g pattern s ten d to b e highl y conserve d i n homologou s protein s eve n thoug h theymaydiverg e consid erabl yi n structur

e an d function , suggestin g that othe rmember s o fth e superfamily , includin g o^-g , posses s a simila rthree-dimensiona l structure . Th e onl ymembe ro

fth e protei n superfamil ywit h a clearl ydefine d physiologica l functio n i s retinol-bindin g protein . More circumstantia l evi denc e suggest s tha

t th e superfamil y member s serv e a s carriers o flipophili c molecule s (Pervai z an d Brew , 1987) . Th e mod e o fbindin g i n whic h th e lipi d ligan d i s enclose d withi n th e B-barre l impresse d Pervai z an d Brewa s not unlik e th e protective rol e o fth e calyxt o a flower . O n thi s basis , the y suggeste d th e illustrativ e name , lipocalins , fo rth e superfamil yo fproteins . Tabl e 2 illustrate s th e informatio n availabl e o n severa l member s o fth e lipocali n superfamily . whic h include s 0L, o-g , retinol-bindin g protein , apolipoprotei n D , a,-aci d glycoprotein , a-microglobulin , ruminan t B-lacto globulin , an d pyrazine-bindin g protei n (i.e. . odorant-bindin g protein) , rat odorant-bindin g protein , an d MUP . Som e o fth e members o

fth e lipocali n superfamily . suc h a s retinol-bindin g protein , a,-aci d glycoprotein , an d a, - microglobuli n have bee n identifie d i n man yspecies , an d thei rpropertie s appea rto b e species-independent , suggestin g that theyshare a commo n vita l functio n (AkerstrO m an d Logdberg , 1990) . Others , suc h a s a^- g an d MU P see m t o b e species-dependent . Severa l function s have bee n suggeste d fo rcu-g . Cavaggion i e t al . (1987 ) speculate d tha i ots- g mayserve t o transfe rodoran t s suc h a s etherea l lipi d pheromone s fro m mal e ra t urin e t o th e ai rfo rattractin g females . Glandula rtissu e productio n o ffiL-g help s suppor

t thes e speculation s (Murtye t al. , 1987 ; Mancin i e t at , 1989) . I n addition , rj^- g ha s bee n identifie d a s a fatt

yacid-bindin g protei n o fth e kidney(Kimura e

t al. , 1989 ) an d mayserve t o transpor t fatt y acid , a n importan t energ ysourc e i n kidney , withi n rena l epithelia l cells . Brook s (1987 ) foun d a protei n structurall y relate d t o o^- g tha

t i s synthesize d an d secrete d by th e ra t epididymi s unde r th e influenc e o

fandrogeni c hormones . H e speculate d tha t th e functio n o

f thes e protein s ma

yb e to carr

yretinoid s withi n th e lume n o fth e mal e reproductive tract . Othe rmembers o

fth e lipocali n superfamily , suc h a s retinol-bindin g protein , apolipoprotei n D , B-lactoglobulin , an d a,-aci d glycoprotein , functio n i n th e transpor

t o flipid s betwee n cell s an d acros s hydrophili c barrier s (Pevsne re t al. , 1988) . Th e lipid s boun d byth e protein s diffe rconsiderabl y i n structure an d rang e fro m odorant s i n ra t nasa l epitheliu m t o huma n cholestero l an d retino l (vitami n A) . It i s no t yet clea rho

wselective thes e protein s ar e fo rspecifi c ligand s o rwhethe ra give n protei n migh t bin d a wid e spectru m o

fsmal l hydrophobi c molecules . Bot h case s migh t occu rsinc e 

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•• : a retinol-bindin g protei n i s quit e specifi c fo rretinol , wherea s odorant-bindin g protein s may hav e a broa d rang e o fligand s (Godovac-Zimmermann , 1988) . Th e abilit

yo fa chemica l t o serve a s a ligan d fo ron e membe rofth e superfamil yappear s t o b e a poo rpredicto ro fbindin g affinit

yfo rothe r members o

fth e superfamily . Cavaggion i e t al . (1990 ) measure d th e bindin g affinities o

fa^-g , M UP , an d pyrazine-bindin g protei n isolate d fro m cal fnasa l mucos a fo ra serie s o

fodorants . MU P boun d onl yon e o fthes e chemicals , pyrazine-bindin g protei n boun d six , an d <*sg boun d twelve . Th e bes t ligan d fo rCLg wa s chemicall yunrelate d to th e best ligand s fo rth e othe rtw o proteins , whic h wer e als o chemicall yunrelated . E. Characteristics

of Alpha^-globulln Alphas-globuli n wa s first characterize d i n mal e rat urin e (Ro yan d Neuhaus . 1967) . Al l isoform s o fa. g ar e anioni c at neutra l p H althoug h the y have varyin g isoelectri c points . Th e molecula rweigh t o fo^- g ha s bee n reporte d t o b e 18.00 0 to 20.00 0 daltons . I n al l ra t strain s teste d t o date , except fo rth e NC I Black-Reite r(NBR ) rat , a strai n tha t appear s t o hav e a tissue - an d gene-specifi c regulator ydefec t involvin g o^ g (Chatterje e e t al. , 1989) , th e majo rurinar ysourc e o

fa_- g i s th e live rwher e a^- g mRN A constitutes approximatel y1 percen t o fin e hepati c mRN A populatio n (Sippe l e t al. , 1976 ; Kurtz an d Feigelson , 1977) . Th e hepati c isoform s ofo^- g ma y var ythroughou t th e lifetim e (Ro ye t al. , 1983) . Synthesi s o fth e protei n i n rat live ri s unde rmuttihormona l control , particularl yandrogen , bu t als o glu - cocorticoids , thyroi d hormones , insuli n an d growt h hormon e (Feigelso n an d Kurtz , 1977 ; Ro yan d Chatterjee , 1983) . Thes e hormone s appea rt o ac t b y regulatin g th e steady-stat e leve l o fa^- g mRN A (Kurt z an d Feigelson , 1977) . Neithe rc^ g no rits correspondin g mRN A ar e detectabl e i n th e liver s of sexuall yintac

t femal e rat s (Sippe l e t al. , 1975,1976 ; Maclnne s e t al. , 1986) . However , a verylo

wbackgroun d leve l o

fth e mRN A ha s bee n indicate d i n th e ovariectomize d femal e ra t (Chatterje e e t al. , 1979) . an d ovariectom yi n concer t wit h androge n treatmen t induce s a paralle l increas e i n o^- g an d it s mRN A i n femal e ra

t live r(Ro yan d Neuhaus . 1967 ; Sippe l e t at. , 1975) . Althoug h plasm a an d urinar ya^-g derive predominantl yfro m th e live r i n mal e rats , hig h level s o fo^- g an d its mRN A ar e als o presen t in th e preputia l glan d ofbot h mal e an d femal e rats , an d neithe rcastratio n no r ovariectom ysignificantl y alte rth e preputia l concentratio n o fthi s protei n an d its mRN A (Murtyet al , 1988) . Alphas-globuli n mRN A ha s als o bee n detecte d i n th e femal e mammaryglan d durin g pregnancy , an d i n th e submaxillary , lachrymal , Meibomian , an d periana l gland s o frat s ofbot h sexe s (Maclnne s e t al. , 1986 ; Mancin i et al. , 1989) . Th e femal e form s o fotsg sho

wdistinc t difference s fro m mal e ra t a.- g suggestin g tha t theyar e encode d bydifferen t gene s (Vandore n e l al. , 1983) . Lo wlevel s o

fa^- g firs

t becom e detectabl e i n th e mal e rat live runde rth e stimulu s o

ftestosteron e at 3 5 t o 4 0 days , reachin g maximu m adul t level s b y 6 0 t o 8 0 day s (Ro yetal. . 1983 ; Motwan i etal. . 1984 ; Maclnne s etal. , 1986) . Du e to th e developmen t o fhepati c insensitivit

yto androge n durin g aging , hepati c synthesi s o

fa 2o - g begin s to fal l graduall yi n mal e rat s som e tim e afte r 1 8 1 95 month s o fage . By2 2 month s o fage . ther e ha s bee n a dro p o fove r9 0 percent , wit h a_- g bein g virtuall yundetectabl e i n senescen t animal s (Ro y e t al. , 1983 ; Motwan i et al. , 1984 ; Richardso n et al. , 1987) . Rena l cortica l tissu e conten t (Murtyet al. , 1988 ) an d urinar yexcretio n (Neuhau s an d Ftory , 1978 ; Motwan i e t al. , 1984 ) o fcts- g reflect th e sam e age-relate d trend s a s synthesi s i n th e liver . I n th e matur e mal e rat , approximatel y5 0 rn g o fo^- g i s filtere d pe rday , 4 0 percen t o

fth e filtere d protei n bein g excrete d i n th e urin e an d 6 0 percen t undergoin g reabsorptio n an d catabolis m (Neuhau s e t al. . 1981 ; Caudil l et al. , 1991) . Alphas-globuli n i s catabolize d slowl yrelative t o mos t othe r protein s i n th e glomerula rfiltrate wit h th e half-lif e i n plasma , kidneycytosol , o rlysosoma l preparation s bein g 5 t o 8 hours (Geertze n et al. , 1973 ; Ekstrom . 1983 ; Lehman-McKeema n et al. , 1990a) . I n vitro studie s indicate that Os- g i s mor e resistan t t o lysosoma l enzym e digestio n tha n bovin e B-lactogtobuli n an d lysozym e (Charbonnea u e t al. , 1988) . I n anothe rstud y comparin g member s o fth e protei n superfamily , o^- g an d a,-aci d glyco protei n wer e th e mos t resistan t t o proteinas e Kdigestio n whil e retinol bindin g protei n an d B-lactoglobuli n wer e 1000 - t o 100,000-fol d more easil y hydrolyse d (Borghof

fe t al. , 1990) . Thes e dat a indicat e that 0L- g mayb e more likel yt o accumulat e i n th e kidneytha n most othe rmember s o fth e superfamil yifshift s i n th e balanc e betwee n reabsorptio n an d hydrolysi s occur . F. Sex and Species

Comparison

of Urinary Protein Content

of the Lipocalin

Superfamily Relative t o th e femal e rat . an d othe rspecie s includin g humans , th e norma l matur e mal e rat i s physiologicall y proteinuric . Thi s i s du e to th e amoun t o

fOL- g secrete d i n mal e rat urine , 1.36-8.6 4 mg/day/ g kidney (Neuhau s an d Lerseth , 1979) , whic h i s 10 0 t o 30 0 time s more tha n observe d i n femal e rat urin e (Shapir o an d Sachchidananda . 1982 ; Vandore n et al. , 1983) . Th e mous e can als o b e describe d a s physiologicall y proteinuri c becaus e o

fa hig h urinar

yconten t o

fMU P (Thung . 1962) . MU P shows th e greates t similarit

yt o rj^- g i n th e lipocali n superfamily , sharin g 9 0 percen t amin o aci d sequenc e homolog y(Dola n e t al. , 1982) . Representin g a grou p o fprotein s encode d b

ya multigen e family , MU P i s synthesize d i n th e live r o fmic e o fbot h sexe s bu t a t rate s fou rto five time s greate ri n male s tha n female s (Hasti e et al. , 1979 ; Ro yan d Chatterjee , 1983) . Dail yurinar y excretio n o

fMU P varie s considerabl yamon g strain s (Szok a an d Pafgen , 1978) . I n th e B6C3F 1 strain , male s hav e bee n show n t o excret e 14. 9 m g o fMUP/da yI n th e urine , an d females , 2. 1 mg/da y (Lehman-McKeema n et al. , 1990b) . Adjuste d fo rbod yweight , a mal e B6C3F 1 mous e therefor e excrete s approximatel y60 0 mg/kg/day o fMUP . som e 12-fol d highe rtha n OL- g urinar

yexcretio n b

yth e mal e rat . Unlik

e th e rat , however , wher e 6 0 percen t o

ffiltere d a, u - g i s reabsorbe d b yth e kidney , MU P i s no t reabsorbe d i n th e mous e an d appear s t o b e totall yexcrete d (Caudil l et al. , 1991) . I n contrast , norma l huma n urin e contain s relativel ylittl e protein , onl y1 percen t o

fth e tota l concentratio n presen t i n matur e mal e ra t urin e (Olso n e t al. , 1990) . Huma n urinar yprotein s are predominantl yhigh-molecularweigh t specie s wit h onl ymino rcomponent s weighin g les s tha n 66.00 0 daltons . Withi n th e low-molecular-weigh t fraction , trac e amounts o fprotein s represen t th e lipocali n superfamily . bu t non e appea rt o share molecula r weigh t identit

ywit h rjt^-g . Th e urinar yexcretio n o

fretinol-bindin g protein , a,-aci d glycoprotei n an d a,-microglobuli n ha s bee n measure d a t 0.000 1 t o 0.0007 , 0.000 6 t o 0.002 . an d 0.0 2 t o 0.0 5 mg/day/ g kidney , respectivel y (Berggard , 1970 ; Peterso n an d Berggard . 1971 ; Ekstro m an d Berggard . 1977) . Thus , th e urinar yexcretio n o

fo^- g i n th e mal e rat i s approximatel y tw o order s o

fmagnitud e greate rtha n th e huma n urinar yconten t o

fth e thre e superfamil yprotein s combined . Recently , a sex-dependen t protei n o funknow n origi n an d function , terme d urin e protei n 1 , wa s identifie d i n norma l huma n urin e (Bernard e t al. . 1989) . Th e molecula rfeature s o fprotei n 1 are simila rt o o^- g a s i t ha s a molecula rweigh t o

fapproximatel y21,00 0 dalton s an d a n isoelectri c poin t aroun d 4. 8 althoug h its amin o acid s hav e not bee n full ysequence d (persona l communication . R . Lauwerys . Catholi c Universityo fLowain , Belgium , t o I . Rodgers , Marc h 25.1991) . Protei n 1 occurs i n bot h sexe s fro m a n earl y age , bu t increase s substantiall yi n male s afte rpuberty , reachin g u p t o a 50-fol d differenc e ove rfemale s durin g lat e adolescence . A 5-fok J mal e t o femal e differentia l persist s throug h adulthood . Averag e urinaryconcen tration s o

fprotei n 1 have bee n determine d a s 10 8 an d 3. 2 ug/ L respectivel y fo rmale s an d female s age d 1 5 t o 2 0 years , an d 24. 7 an d 5. 8 ug/ L fo rmale s an d female s i n th e 2 0 t o 6 0 yea rag e rang e (Bernar d et al. , 1989) . Suc h level s o fprotein l i n huma n mal e urine , however , are calculate d a s fou rto fiv e order s o

fmagnitud e les s tha n o^- g concentration s i n th e urin e o fmal e rats . G . Noncovalent

Binding

to Alphas-globulin

and Its Homologues I t ha s bee n suggeste d tha t CIG A bin d reversibl yan d noncovalentl yt o OL^-g , formin g a resultan t comple xthat i s eve n more poorl ydigeste d i n th e mal e rat kidneytha n a^- g (Swenberg , 1989) . 1. Chemical

entities bound to alphas-globulin I n a fe winstances , th e specifi c chemica l entitycomplexe d wit h cr^- g ha s bee n identified . TMP , a branche d chai n aliphati c hydrocarbo n presen t i n gasolin e wa s th e first mode l CIG A t o b e studie d i n thi s manner . Whe n ("Cj TM P wa s administere d i n a singl e ora l dos e to rats , radioactivitywa s retaine d i n th e kidneys o fmales , bu t not o

ffemale s (Klos s e t al. . 1985 ; Charbonnea u e t al. , 1987) . Th e majo rmetabolit e o fTM P i n th e mal e rat kidney s wa s identifie d a s 2,4.4-trimethyl-2-pentano l (T M POH) (Charbonnea u et al. , 1987) . I n a separat e report , TMPO H wa s show n t o b e th e onl yligan d 'o rot^- g wheneve rTM P wa s administere d t o th e mal e rat (Loc k e t al. , 1987a) . TMPO H wa s no t detecte d i n th e kidneytissu e o fth e femal e rats , whic h excrete d more conjugate d TMPO H (glucuronide s an d sulfates ) tha n th e male s (Charbonnea u e t al. , 1987) . Late rstudie s confirmed , a s sus pected , that th e TMPOH-ots- g complexi s cleare d slowl yfro m mal e ra t kidney(Swenberg . 1989) .ro r U-IMIIUIWIIV , U-MHICMIWIIW-I.^-UXHJ O na s oee n snow n 1 0 ut» ui w predominan t metabolite bindin g t o o^- g althoug h d-limonen e als o bind s t o som e exten t (Lehman-McKeema n e l al. , 1989) . Fo risophorone , th e ligan d i s th e paren t compoun d (Strasse ret al. , 1988) . Followin g exposure o fth e mal e ra t to 1,4-DCB , bot h th e paren t chemica l an d th e metabolite , 2,5 - dichlorophenol . boun d t o 0L- g (Charbonnea u e t al. , 1989) . Abou t 4 0 percen t o

fth e 3.5,5-trimethythexanoyloxybenzen e sulfonat e administere d t o mal e rats boun d t o kidne yprotein s eve n thoug h n o protei n bindin g wa s observe d i n th e mous e o rfemal e ra t kidney(Lehman-McKeema n e t al. , 1991) . Fou rmetabolite s wer e identifie d i n th e 0L- g protei n fraction , th e mai n componen t bein g th e gamma-lacton e o f3.5.5-trimethy l hexanoi c acid . 2. Nature

of the association Th e natur

e o fth e associatio n o

fCIG A wit h o^- g wa s explore d initiall y b yLoc k et al . (1987a ) wh o dose d sexuall ymature mal e Fische r34 4 (F344 ) rats wit h PHJ-TMP , kilte d the m 8 t o 7 2 hours later , an d homogenize d th e kidneys . Cytosol , obtaine d b ycentrifugatio n o fth e homogenat e a t 116,00 0 g . wa s applie d t o a Sephade xG-7 5 column . Abou t 2 6 percen t o fth e cytoso l radiolabe l (15 % ofal l radiolabe l i n th e kidney) edite d i n th e fractio n containin g a^-g . Approximatel y1 9 percen t o fth e radiolabe l i n th e cytoso l wa s nondialyzabl e followin g overnigh t equilibriu m dialysi s agains t phos phat e buffer . Chromatograph yo fth e diaryze d cytoso l showe d tha t th e nondialyzabl e radiolabele d materia l coelute d wit h th e pea k containin g 

## f -g . Whe n 0. 1 percen t sodiu m dodecy l sulfate (SDS) , a detergen t whic h act s th e secondar

yan d tertiarystructure o fproteins , wa s adde d t o th e dialysi s buffer , ther e wa s a significan t los s o fbinding . Thes e result s suggeste d a reversibl e bindin g betwee n th e TM P metabolit e an d th e protei n fractio n containin g o^- g (Loc k et al. , 1987a) . Th e reversibility o fchemica l bindin g wit h a_-g , whethe rparen t compoun d o rmetabolite , ha s bee n confirme d wit h isophoron e (Strasse re t al. , 1988) , 1,4-DC B (Charbonnea u e t al. , 1989) , d-limonen e (Lehman-McKeema n e t al. , 1989) , 3,5,5 - trimethylhexanoioxybenzen e sulfonat e (Lehman-McKeema n e t al. , 1991) , an d lindan e (Dietric h an d Swenberg , 1991a) . I n th e d-limonen e stud y(Lehman-McKeema n e t al. , 1989) , th e amoun t o fradioactivit

yobserve d i n th e kidney s o fSprague-Dawle yrat s 2 4 hours afte rora l administratio n o fP 4 C]-d-limonen e wa s abou t 2. 5 time s highe ri n male s tha n i n females . Equitibriu m dialysis , i n th e presenc e o rabsenc e o f SDS , indicate d that approximatel y4 0 percen t o fth e radioactive materia l retaine d i n th e mal e rat kidne ywa s associate d wit h protein s i n a reverstol e manner . Ge l filtratio n high-performanc e liqui d chromatograph y(HPLC) , reverse-phas e HPLC , an d amin o aci d sequencin g demonstrate d that thi s radioactive materia l wa s associate d wit h OL-Q. N O d-limonen e o r d-limonen e metabolite coelute d wit h femal e rat kidney proteins . I n th e 3,5,5 - trimethylhexanoloxybenzen e sulfonat e stud y(Lehman-McKeema n e l al. , 1991) , distributio n o

fth e chemica l wa s examine d i n mic e an d rat s o fbot h sexes . Th e mal e rat kidneycontaine d roughl y1 0 time s th e concentratio n o fchemica l a s th e femal e rat kidney , an d th e concentration s i n mous e kidneywere eve n lowe rtha n thos e i n th e femal e rat . 3. Binding ot CIGA to other macromoiecuies Reversibl e bindin g generall yimplie s a dissociabl e chemical-protei n interactio n i n whic h th e fre e chemica l ca n b e liberate d fro m th e protei n without havin g produce d molecula rdamage . I n contrast , i n c o vaten t bindin g a reactive chemica l species , usuall y a n electrophile , react s wit h nucleophili c centers i n target molecule s comprisin g enzymes , othe rproteins , nuclei c acids , o rlipids . CIG A appea rt o diffe rfro m man yknow n chemica l toxins , nephrotoxin s included , whic h bin d covalentl yan d irreversibl yt o protein s and/o rDN A an d throug h thi s proces s caus e cellula rinjury . A DN A bindin g stud ywit h F34 4 rat s an d B6C3F 1 mic e o fbot h sexe s wa s performe d usin g [1,3,5,- 14 C]-isophoron e (Thie ret al. , 1990) . Twenty-fou r hour s afte rth e animal s wer e administere d a 50 0 m g dos e bygavage . live r an d kidneys wer e processe d fo rdeterminatio n o fDN A binding . Neithe r isophoron e no rit s metabolite s showe d covalen t bindin g t o DNA . I n addition . metaboVcall yforme d degradatio n products wer e not incorporate d int o th e DN A b y d & ngyo . synthesi s o

fDN A fro m labele d fragment s o fth e xenobiotic . I n contrast t o 1,4-DCB , whic h i s a CIGA , 1,2-DCB , a closel yrelate d isomer , doe s no

t induc e hyalin e droplet s an d appear s t o bin d covalentl yt o protein s i n th e mal e rat liver , plasma , an d kidney(Charbonnea u et al. , 1989) . Whe n administere d orall yt o mal e rats , 1,4-DC B (an d its metabolit e 2,5 - dichlorophenol ) i n th e kidneycytoso l elute d a s a singl e pea k i n th e low

molecular-weigh t fractio n containin g o_-g . Dialysi s o fth e kidne ycytoso l wit h SD S le d t o a substantia l toss o f1,4-DCB , demonstratin g th e reversibl e natur e o fth e CIGA-protei n binding . 1,2-DC B boun d t o low-molecular weigh t protein s i n th e kidneycytoso l o fmal e rats , bu t i t als o boun d t o protein s i n th e 64,00 0 t o 70,00 0 dalto n range . Dialysi s o fth e kidne ycytoso l wit h SD S faile d t o remove approximatel yhal fth e 1,2-DCB , suggestin g substantia l covalen t bindin g o

fthi s chemica l i n th e mal e rat kidney . 4. Specificity

ot the interaction

of CIGA with

alphas-globulin Th e capacityo

fCIG A t o serve a s ligand s fo rothe rlipocalins , som e o f whic h ar e foun d i n humans , ha s bee n investigated . Preliminarystudie s designe d t o determin e th e accumulatin g protei n i n th e kidne yo fmal e rat s expose d t o decali n employe d two-dimensiona l ge l electrophoresi s o fra t kidne yhomogenat e (Alde n et al. . 1984) . Althoug h othe rprotein s i n th e lipocali n superfamil yare presen t i n th e mal e rat , decali n wa s associate d solel ywit h ot^-g . Othe rpreliminar ystudie s involvin g th e i n vitro bindin g o f TMPO H t o lipocalin s suggest that TMPOH . whic h bind s reversibf y t o OL- g i n vitro , mayals o bin d reversrbl y to thre e othe rmember s ofth e superfamily , i.e. , retinol-bindin g protein . a,-aci d glycoprotei n an d B-lactog(obulin(Borghof f e t al. . 1988) . TMPO H di d no t bin d to B 2 -microglobuli n o rlysozyme . lowmolecular-weigh t protein s tha t are no t member s o

fth e superfamily . D-limonene-1,2-oxid e als o doe s no t appea rt o bin d t o a,-aci d glycoprotei n o rurin e protei n 1 i n i n vitro studie s (persona l communication , L . Lehman McKeeman , Procte ran d Gamble , to I . Rodgers , February27,1991) . Ga s chromatographi c analysi s i n experiment s wit h live rmicrosome s have show n tha

t mice ar e abl e t o oxidize d-limonen e t o cis-d-limonene-1,2 - 2 2 2 3oxide , a s i n th e rat , althoug h som e qualitative an d quantitative specie s

difference s wer e note d (Lehman-McKeeman , 1990b) . However , equilib rium saturatio n bindin g studie s di d no t demonstrat e an y interactio n betwee n d-limonen e o rits metabolite s an d MU P i n mal e o rfemal e mic e (Caudil l e t al. , 1991 ; Lehman-McKeeman , 1990b) . Thes e result s ad d furthe rsuppor t t o th e specificit yo fth e interactio n betwee n CIG A an d a^-g . Whe n PHj-retino l wa s administere d t o mal e rats , retinol - derive d radio activitycoelute d wit h th e protei n fractio n i n cytoso l containin g OL-g . How ever , retino l di d not produc e accumulatio n o fhyalin e droplet s o rots- g (Borghof fe t al. , 1989) . I n vitro studie s o n th e bindin g affinitie s ofretino l an d severa l CIG A fo r0L- g sho wthat retino l ca n compet e wit h CIG A fo rbindin g t o rjts- g (Borghof

fe Tal. , 1991) . Thes e studie s suggest that hyalin e droplet accumulatio n ma y no

t depen d o n ho wstrongl ya chemica l bind s t o o^-g , bu t o n whethe rth e chemica l cause s a conformationa l chang e i n th e protei n that tnhbrt s protei n catabolis m (Borghof fe t al. , 1990) . Bindin g affinitie s measure d i n i n vitr o studie s generall yhav e not correlate d wel l wit h th e efficacyo fchemical s fo rcausin g hyalin e droplet accumulation . Othe rfactors affectin g th e developmen t o fhyalin e droplet accumulatio n ar e th e concentratio n o fth e CIGA-protei n comple xi n th e tubul e lumen , th e rat e ofbreakdow n ofCIGA-protei n complexe s i n th e tubul e cells , th e deat h ofcell s resultin g fro m abnorma l accumulatio n o f hyalin e droplets , an d th e subsequen t appearanc e o fcel l debri s i n th e lume n o ftubul e cells . Thes e factor s ar e discusse d i n th e followin g sections . H. Catabolism

of Alphas-globulin

Complexed

With CIGA Reduce d rena l lysosoma l catabolis m o fth e CIGA-OL- g complexlead s t o its accumulatio n i n th e cell s o fth e proxima l rena l tubule , causin g lysosoma l protei n overloa d an d individua l cel l deat h (Swenber g e t al. , 1989) . Figure 3 illustrate s thi s propose d sequenc e o fevents . Lysosoma l degradatio n o fots- g boun d t o CIGA ha s bee n studie d b y measurin g th e digestio n rat e o fth e protei n recovere d fro m treate d mal e ra t kidne y(Charbonnea u e t al. , 1988 ) o ro fpurifie d urine-derive d protei n conjugate d wit h CIG A i n vitr o (Lehman-McKeema n e t al. , 1990a) . Charbonnea u e t al . (1988 ) foun d tha t bot h proteinas e Ko ra mixture o f standar d proteas e enzyme s o fnon-rat origi n digeste d o^- g fro m rat s treate d wit h TM P a t a muc h slowe rrat e tha n ots- g fro m untreate d rats . Usin g a n i n vitro incubatio n syste m wit h rena l cortexlysosome s pre pare d fro m mal e rats , Lehman-McKeema n e t al . (1990b ) demonstrate d that th e reversibl e bindin g o fd-limonene . 1,4-DCB , an d isophoron e o rthei r metabolite s impaire d th e degradatio n o fo^- g byone-third . Unde rth e experimenta l condition s employed , thi s wa s equivalen t t o a n extensio n o f th e apparen t half-life o

fot^- g fro m 6.6 7 t o 1 0 hours . Th e stud yi s particularl y interestin g becaus e it shows tha t reversibl e bindin g o fa CIG A t o o^- g doe s no t necessaril yalte rth e rat e o fprotei n degradation , bu t that thi s ma yb e a functio n o

fa metabolite . Thus , d-limonen e an d 1,4-DC B di d no t impai r hydrolysi s o

fou^- g bu t thei rrespective boun d metabolites , d-limonene-1,2 - Tubul e Lume n Atohs*,-Qtobul n + CtO A



### Bloo d Proxima l (P2 ) Tubul e Cel l

Flgw S: Schematic

representation

ot the uptake and fate of alphas-globulin complexed

with at CfGA in hydrocarbon

nephrotoxicity. Source: Modification

of Figure 2. above.

### oxide , an d 2,5-dichtorophenol , did

. Wit h isophorone

. however , i t wa s th e

paren t compoun d aton e whic h produce d th e effect

. Thi s apparen t nee d t o reduce protei n degradatio n migh t offe ra n explanatio n t o describ e wh y chemicals , suc h a s retinol , have bee n show n t o bin d t o OL^-g

. withou t producin g hyalin e drople t accumulation

. Administratio n o fleupepti n (a n inhibito ro fth e lysosoma l peptidase , cathepsi n B ) cause d a rapi d OL- g accumulatio n i n th e kidney , indistinguish abl e fro m tha t induce d b yTM P o rgasolin e (Olso n e t al. , 1988)

. Thes e variou s observation s provid e evidence tha t CIGA-induce d hyalin e drople t accumulatio n ma

yresul t fro m a reduce d protei n degradatio n rat e eithe rby (1 ) makin g th e protei n harde rt o diges t o r(2 ) inhbHin g enzymati ccompo nents o fth e proteolyti cprocess . Studie s b yCharbonnea u e t al

. (1988 ) an d Lehman-McKeema n et al

. (1990a ) suppor t th e forme rb yindicatin g tha t th e TM P metabolite-protei n complexi s more resistan t t o hydrolysi s tha n fre e Os-g

. Furthermore , Murtye t al

. (1988 ) foun d that unleade d gasolin e wa s not associate d wit h a reduction , bu t rathe ra n increase , i n rat kidne ylysosoma l proteolyti cenzym e activity

. / . Structure-Activity

Relationships

for CIGA A n abilityt o predict thos e chemical s that wil l induc e accumulatio n o f OL- g i n th e mal e ra t throug h structura l relationship s woul d b e clearl y advantageous

. Th e fac t tha t relativel ymino rmetabolite s suc h a s d-limonene-1,2-epoxid e ca n accoun t torth e majorityofth e associatio n wit h ots-g , however , restrict s th e presen t utilityofstructur e activitycalculation s a s a predictiv e tool

. Nevertheless , som e association s hav e bee n observed

. Lehman-McKeema n e t al

. (1990a ) note d tha t retarde d degradatio n o f Os- g correlate s wit h th e presenc e o n th e activ e CIG Ao rmetabolit e ofa n oxyge n functio n o

fon e typ e o ranother , i.e. , a n hydroxy l grou p fo rTMPO H an d 2,5-dichtorophenol , a n epoxid e fo rd-limonene-1,2-oxide , an d a keton e functio n fo risophorone

. Anothe rrecen t stud yemploye d a quantitativ e approac h t o determin e th e structura l feature s necessar yt o induc e excessiv e hyalin e drople t activity i n mal e rat s (Bomhard e t al. , 1990)

. Base d o n dat a fo ra numbe rofligh t hydrocarbons , Bomhard et al

. surmise d tha t a n n-octanol/wate rpartitio n coefficien t above 3. 5 an d th e presence ofa n isopenty l structura l moietyare associate d wit h increase d hyalin e drople t formatio n i n mal e rats

. Abindin g sit e mode l fo raliphatic s wa s derive d fro m thi s information

. Th e mode l wa s the n generalize d t o includ e cycloaliphatic s bysubstitutin g th e requiremen t fo ra n isopenty l structur e wit h a requiremen t fo rth e presenc e ofat leas t on e tertiar ycarbo n atom

. Usin g thi s bindin g sit e model , Bomhard e t al

. predicte d th e hyaline-drople t inducin g activit yo f1 8 previousl yunteste d hydrocarbons

. Thes e chemical s wer e the n teste d fo rabilityt o induc e hyalin e drople t accumulatio n i n adul t mal e Wista rrats

. Eve n thoug h th e bindin g sit e mode l wa s base d o n th e structur e o fth e paren t compoun d an d di d not allo wfo r active metabolites , th e result s i n th e rat s wer e describe d a s bein g i n goo d agreemen t wit h th e predictions

.

2 6 

### Borghof fet al

. (1991 ) determine d th e apparen t bindin g affinit yt o oL u 

### - g fo ra numbe rofchemical s associate d wit h 0L u 

### - g nephropath yan d measure d thei rabilityt o compet e wit h TMPOH

. Usin g molecula rmodelin g an d informatio n o n th e most active compounds , thes e investigators conclude d that th e presenc e ofa n electronegativ e atom fo rhydroge n bondin g i s a critica l facto ri n determinin g bindin g affinity

. Lipophilicityals o seeme d crucia l fo rhydrophobi cinteractions , bu t th e presenc e o fa n electronegativ e ato m wa s necessar

yfo rgreate ractivity

. Steri cvolum e wa s als o considere d t o playa n essentia l rol e i n bindin g activity

. Th e conclusion s o

fBorghof fe t al

. (1991 ) are consisten t wit h th e notio n that OL^- g i s capabl e o

ftransportin g lipophili ccompound s withi n a bindin g sit e pocke t o

fspecifi cdimensions

. Sinc e bindin g affinitydoe s not correlat e wel l wit h hyalin e drople t formation , however , th e abilityo fthi s structura l featur e t o serve a s a predictiv e too l woul d appea rlimited

. IV . Alphas-globuli n Nephropath y Substance s reporte d to induc e increase d formatio n o fhyalin e droplet s i n proxima l tubul e cell s o fmal e rat s are liste d i n Tabl e A-1 , alon g wit h availabl e informatio n o n whethe rth e accumulatin g protei n i s o^-g

. Th e nephrotoxicitythat ca n ensu e fro m hyalin e drople t accumulatio n i s nove l becaus e i t i s associate d wit h excessiv e ots- g accumulation

. Thi s o^- g accumulatio n i s believe d t o initiat e a sequenc e o fevent s resultin g i n chroni c proliferatio n oftubul e epithelium , a s wel l a s a n exacerbatio n o fCPN

. Becaus e a 2u 

### - g i s a mal e rat-specifi cprotein , nephropath yinduce d b y accumulatio n o

fOL- g woul d not b e expecte d t o occu ri n femal e rats , mic e o feithe rsex , o rothe rspecies

. Th e propose d sequence o fhistopathologica l change s i s base d mainl y o n researc h studie s wit h fou rmode l substances , unleade d gasolin e an d TM P (Shor

t e t al. , 1986 ; 1987 ; 1989a)

. decali n (Alde n e t al. , 1984 ; Kanerv a etal. , 1987a,b,c ; Ston e etal. , 1987) , an d d-limonen e (Kanerv a etal. , 1987b ; Web b et al. , 1989)

. Fo reve n thes e fou rsubstances , not al l ofth e individua l lesion s i n th e propose d progressio n hav e bee n show n t o belon g t o a sequence o

finterrelate d events

. Specifi cinformatio n pertainin g t o lesio n nature an d sequenc e i s lackin g fo rman yo fth e hyaline-drople t inducers liste d i n Tabl e A-1

. Muc h o fth e informatio n usefu l fo rdefinin g th e pathologi csequela e t o ots- g accumulatio n doe s no t require chroni cexposure . Accumulatio n of OL- g i s visibl e withi n a matte rofday s an d th e respons e t o chroni c administratio n ofCIG Amigh t eve n diminis h sinc e o_- g level s declin e i n agin g mal e rat s (Murt

ye t al.

. 1988)

. Th e nephrotoxicityassociate d wit h o_- g accumulatio n migh t als o b e influence d b yage . Certainly , th e age relate d progressio n o

fCP N obscure s th e lesion s directl yrelate d t o CIG A administration , makin g evaluatio n o fth e chroni csequenc e o flesion s especiall ydifficult

. A. Pathologic

Features

of Alphas-globulin

Nephropathy Rena l lesion s describe d i n scientifi cstudie s a s bein g associate d wit h ots- g nephropath yar

e liste d i n Tabl e 3

. Th e firs t morphologica l manifesta 27Table 3. Summary

of the Histopathology

and Lesion Progression

Reported

in Alpha^ Globulin-Associated Nephrotoxicity 1 . Excessive accumulatio n o l hyalin e droplet s i n th e P 2 segmen t ofth e proxima l tubul e regio n o l kidneyoccurs afte r1 o r2 days . Thi s i s reversibl e withi n 3 day s to 2 week s afte r exposure ceases . 2 . Evidenc e o l single-cel l necrosi s i n th e P 2 segmen t epitheliu m an d exfoliatio n afte r5 day s o l continuou s exposure . 3 . Accumulatio n o l granula rcast s forme d fro m th e cellula rdebri s an d subsequen t tubul e dilation , a t th e junctio n o l th e P 3 segmen t an d th e thi n loo p o l Henle . followin g 2 0 t o 4 0 day s o fcontinuou s exposure . Granula rcast s hav e bee n observe d a t 3 to 1 3 week s afte r commencin g exposur e an d sometime s beyond , u p to 2 years . 4 . Increas e i n ceS proliferatio n withi n th e P 2 segmen t followin g 3 week s ofcontinuou s exposure , remaining elevate d abov e norma l a t 4 8 weeks ot exposure . 5 . Linea rmineralizatio n o ftubule s withi n th e rena l papilla , appearin g betwee n 3 an d 1 2 month s afte ra 28-da yexposure , an d sometimes observe d a t th e en d o fa 2 yea rstudy . 6 . Hyperplasi a o l th e rena l pelvi c epithelia l linin g observe d aroun d 1 year . • 7 . Exacerbatio n ot th e spontaneou s chroni c progressiv e nephropath ysyndrom e commo n i n agin g rats . * 8 . Formatio n o

foccasiona l hyperplasti c loc i withi n cortica l epitheliu m a t chroni c time-points .

' Indirect consequenc e o l progressio n o l lesions .

tio n o l Os- g nephropath yi s th e rapi d accumulatio n o fhyalin e droplet s i n proxima l tubul e cells , developin g withi n 2 4 hour s o fdosin g wit h som e compound s (Web b e

t al. , 1989) . Th e droplet s stai n positivel ywit h Maltory' s HekJenhai n stai n bu t ar e negative fo rperiodi c aci d Schiff , indicatin g thei rprotei n compositio n (Alde n e t al. , 1984) . Maltory' s HekJenhai n stai n i s therefore mor e usefu l tha n conventiona l hematoxyli n an d eosi n ( H & E ) fo rvisualizin g an d quantitatin g th e droplets . As the yrepresen t rysosome-derive d entities , th e droplet s ar e strongl yautoftuorescen t (yellow) i n paraffi n section s unde rultraviolet illumi natio n (unpublishe d observations , G. C Hard) . I n plastic-embedde d tissue , hyalin e droplet s ca n b e visualize d easil ywit h Lee' s methylen e blu e basi c fuschr n (Shor

t e t al. . 1986) . Excessive hyalin e droplet formatio n occur s primaril yi n cell s o

fth e P 2 segment , bu t smal l increase s i n th e numbe ro i hyalin e droplet s ma yals o b e see n i n th e P 1 an d P 3 cell s (Shor t et al. , 1987) . B yligh t microscopi c immunohistochemistry , 0L u - g ha s bee n clearl yan d specificall y localize d t o th e hyalin e droplet s withi n proxima l tubule s (Burnet t e t al. , 1989) . Ultrastruc turally , th e hyalin e droplet s ar e enlarge d secondar ylysosome s partiall y compose d o

fa. g (Gar g et al. , 1989a) . Man yar e pol y angula ro rirregula r i n shape , containin g a condense d crystallin e core suggestive o faggregate d protei n i n pure form . Althoug h th e oL^-g-associated hyalin e droplet accumu latio n persist s durin g chroni c exposure , th e severit yappear s t o lesse n wit h increasin g duratio n o

fexposure (Shor

t etal. , 1989a) . Thi s apparen t wanin g o l th e respons e wit h continue d exposure coul d b e relate d t o declinin g o^- g productio n b

yth e mal e ra

t beginnin g a

t som e stag e alte r5 month s o f ag e (Ro yet al. , 1983 ; Motwan i et al. . 1984 ; Richardso n e t al. , 1987) . Wit h continue d exposure , th e initia l accumulatio n o fOs-g-containin g hyalin e droplet s ma yb e followe d b ya sequenc e o

finterrelate d pathologica l events . (1 ) Scattere d single-cel l necrosi s occur s predominantl yi n th e P 2 segmen t cell s (Short e t al. , 1987 ) wit h subsequen t exfoliatio n ofthes e degenerat e cell s an d cel l fragment s lade n wit h crystalloi d phagolysosome s into th e tubul e lumen . Wit h decalin . a minima l degre e o fcel l degeneration / necrosi s wa s reporte d to b e presen t i n th e proxima l convolute d tubule s afte r 5 day s o

fexposure , becomin g maxima l at 1 9 days , bu t revertin g t o th e minima l leve l afte r3 1 day s o fexposure (Kanerv a e t al. , 1987a) . Scattere d exfoliatio n o

fdroplet-affecte d cell s wa s observe d wit h u p t o 4 8 week s o f exposure to unleade d gasolin e o rTM P (Shor t et al. , 1989a) , indicatin g sustaine d single-cel l los s whil e exposure t o CIG A continues . (2 ) Epithelia l cel l proliferatio n primaril yinvolvin g th e P 2 segmen t oc curs a s a regenerativ e respons e t o cel l damag e an d loss . Thi s ca n b e see n a s increase d number s o fmitoti c figure s o rdemonstrate d bylabelin g technique s fo rDNA-syntheti c activity . Increase d proliferative activityha s bee n recorde d afte ronl ythre e weeks o fpetroleu m hydrocarbo n exposur e (Shor t e t al. , 1987 ) bu t it persiste d durin g 4 8 week s o fchroni c exposur e (Short et al. . 1989a) . (3 ) Granula rcast s compose d o fsloughe d cel l debri s accumulat e at th e junctio n betwee n th e P 3 segmen t o fth e proxima l tubul e an d th e descendin g thi n loo p o

fHenle , that is , at th e junctio n betwee n th e inne ran d oute rstripe s o foute rmedulla , wit h consequen t tubul e dilatio n a t thi s par t o fth e nephro n (Alde n et al. , 1984) . Thi s ca n occu ra s earl ya s tw o t o thre e week s afte rinitia l exposure (Alde n e

t al. , 1984 ; Kanerv a et al. , 1987a) . A s wel l a s comprisin g recognizabl e cel l debris , th e granula rcast s slai n positivel yfo rOL^ g (per sona l communication , J.R . Foster , Centra l Toxicolog yLaboratory , ICI . Macclesfield , t o G . Hard , Novembe r1990 ) indicatin g probabl e derivatio n o f th e debri s fro m cell s whic h ha d accumulate d thi s protein . Granula rcas t formatio n appear s t o b e associate d wit h highe rdose s o l a compoun d rathe r tha n wit h th e lowest dose s that can induc e increase d hyalin e droplet accumulation . A n absenc e o fcast s afte rtreatmen t migh t therefor e reflect a dose-relate d decreas e i n th e severityo fcel l necrosi s an d exfoliatio n (Shor t e t al. , 1986.1987) . (4 ) A t chroni c timepoints , linea rmineralizatio n develop s i n th e rena l papilla , outlinin g affecte d medullar ytubules , alon g wit h hyperplasi a o fth e pelvi c epithelia l linin g (urothelium ) (Alden , 1989) . Wit h unleade d gasoline , thi s lesio n wa s first observe d a t 6 month s o

fexposur e (USEPA . 1987) . Th e mineralizatio n appear s t o for m withi n th e loop s o fHenl e an d ha s bee n identifie d a s calciu m hydroxyapatit e (Trum p e t al. , 1984a) . Th e relationshi p betwee n papillarymineralizatio n an d th e proxima l tubul e lesio n remain s undetermine d bu t th e medullar ylesio n i s presume d to represen t mineral -ize d remnant s o fdebri s fro m disintegratin g granula rcast s that lodg e i n th e preben d segment s o

fth e loops o

fHenl e (Bruner , 1984 , Alden , 1989) . I n turn , urothelia l hyperplasia , whic h mainl yaffect s th e surfac e o fth e rena l papilla , ma yb e a respons e o l th e rena l pelvi s linin g t o papillar ymineraliza tio n (Bruner , 1984 ; Alde n an d Frith . 1991) . B. Rat Urine Chemistry

and CIGA Severa l studie s have examine d rena l functio n i n rat s treate d wit h CIG A an d subsequentl ydevelopin g o^i ) nephropathy . Tw o day s o ftreatmen t wit h TM P resulte d i n mil d urinar yincreas e i n th e lysosoma l enzym e N acety l B-gtucosaminidas e (NAG ) an d alkalin e phosphatase , a decreas e i n creatinine , an d mil d increas e i n urinar ycel l debris . Othe rparameters , aspartat e aminotransferas e (AAT) , urin e osmolality , an d volume , wer e no t affecte d (Fowli e e t al. , 1987) . A single , ora l dos e o fTM P ha d n o effect o n rena l functio n (Stonar d e t al. , 1986) . I n a 14-da ystud ywit h decalin , o fsi x urinar yenzyme s tested , onl yAAT , lactat e dehydrogenase , an d NA G wer e altere d (increases ) a

t day s 2 1 and/o r2 8 (Evan s an d Morgan , 1986) . Simila r results wer e obtaine d to rlevamisol e except that AA Tremaine d norma l (Evan s e t al. , 1988) . Durin g prolonge d treatmen t wit h C 10 -C „ isoparaffini c solvent , u p t o 8 weeks , th e onl yurinar ychange s observe d wer e mil d elevation s o l glucos e an d albumin , slightl ydecrease d concentratin g powe r an d osmolality , an d epithelia l cel l debri s i n th e urine . Ther e wa s n o alteratio n i n urinar yB2 microglobuli n conten t (Phillip s an d Egan , 1984) . Take n together , thes e studie s suggest that CIG A produc e minima l change s i n urinar

ychemistr

yan d ver

ylittl e o rn o glomerula rdysfunctio n o r damage . Th e mino ralteration s see n i n urin e compositio n i n th e day s followin g administratio n o fCIG A suggest als o that hyalin e droplet accumu latio n i s not relate d t o increase d passag e o fseru m protein s by th e glomeru lus . Th e mil d tubul e toxicit yidentifie d b

y clinica l chemistr

yi s a characteristi c o fCIGA . whic h contrast s wit h th e obviou s urinar ychange s associate d wit h th e nephrotoxicit

yinduce d b

ysuc h classica l rena l toxin s a s mercuri c chloride , hexachlorobutadiene , aminoglycosides , an d papillotoxi c agent s (Stonard , 1987) . C . Species

Variation

In the Renal Response

to CIGA Th e male-specifi c effect s o fhyaline-droplet inducers hav e bee n demon strate d ove ra rang e o

frat strain s includin g F344 , Sprague-Dawley . Buffalo , an d Brow n Norwa yrats (Ridde re t al. . 1990) . Hyalin e droplet accumulatio n o rth e spectru m o

flesion s comprisin g o^ g nephropath yhave no t bee n observe d i n femal e rat s o rmice o feithe rsex , followin g treatmen t wit h thes e chemical s (Alde n et al. , 1984 ; Swenber g e t al. . 1989) . I n additio n to thes e studies , othe rhyaline-droplet inducers hav e bee n teste d fo rtoxicit yi n hamster s (jet fuels) , guine a pig s (decalin) , dog s (decalin . jet fuels , d-limonene . an d methy l isobuty l ketone) , an d monkey s (gasolin e an d methy l isobuty l ketone) . N o rena l patholog ywa s demonstrate d i n thes e specie s a

t dose s know n to caus e nephropath yi n mal e rat s (Alde n et al. , 1984 ; Kun a an d Ulrich , 1984 ; MacFartand . 1984 ; MacNaughto n an d Uddin , 1984 ; Phillip s e

t al. , 1987 ) except fo ron e repor t o l mino rchange s i n dog s treate d lo r6 month s wit h d-limonen e (Tsuj i e t al. . 1975) . I n thi s chroni c studya n increase d incidence o fproteinaceou s cast s wa s observe d i n mal e an d femal e beagles , bu t n o tubul e epitheliu m changes , tubul e lume n dilation , o rmineralizatio n wa s observed . However , Web b et al . (1990 ) wer e unabl e t o demonstrat e an yrena l patholog yi n dog s afte r6 month s o f d-limonen e treatmen t a t comparabl e dos e levels . Th e highest dosag e teste d i n th e dogs . 1. 2 mg/kg (Web b et al. . 1990 ; Tsuj i e t al. . 1975) . i s mor e tha n te n time s th e dose s that hav e cause d fran k nephropath yi n mal e rats . Knowledg e concernin g rena l effect s o fCIG A i n human s i s hampere d b y th e lac k o fdat a o n specifi c chemical s i n thi s category , an d th e limitation s impose d bya multiplicit yo ftype s o

foccupationa l an d non-occupationa l exposures . Cas e studie s hav e reporte d a lin k betwee n chroni c rena l diseas e wit h gasoline , solvents , an d jet an d diese l fuel s includin g rar e case s o facut e tubula rnecrosi s (proxima l an d dista l tubul e epithelium ) followin g sever e exposure t o petroleu m distillate s (e.g. , Barriento s et al. , 1977 ; Cris p e t al. . 1979) . Cas e report s cannot b e use d t o establis h a causa l relationshi p bu t mayserv e t o initiate forma l epidemiologi c investigatio n (Churchil l e t al. . 1983) . Epidemiologi c studie s concernin g nonneoplasti c kidneydiseas e an d occupationa l exposure t o hydrocarbon s an d solvent s hav e bee n conducte d onl ysinc e 197 5 (reviewe d b y Askergren , 1986 ; Daniel l et al. , 1988 ; Phillip s e t al. , 1988) . A majorit yo fthes e studie s hav e indicate d a n associatio n betwee n glomerulonephriti s an d exposur e t o hydrocarbons , especiall y organi c solvent s o rgasoline . Som e hav e suggeste d a positive associatio n betwee n th e presenc e o fglomerula rdiseas e an d duratio n an d severityo f occupationa l exposur

e to hydrocarbo n solvents , includin g tetrachloroethylen e whic h i s a CIGA i n mal e rats (Kluwe e t al. , 1984) . However , man yo fth e earlie rstudie s ar e considere d t o b e methodologicall ylimite d (Churchil l e t al. , 1983 ; Askergren , 1986 ; Phillips etal. , 1988) . Majo rshortcoming s hav e bee n heterogeneou s cas e definition , us e ofinappropriat e contro l group s o r nonblinde d interviewers , an d failur e to conside rrecal l bia s o rt o adequatel y defin e hydrocarbo n exposure (Phillip s e t al. , 1988) . Mor e recently . Steenlan d e t al . (1990) , investigatin g specifi c occupa tiona l exposure s associate d wit h end-stag e rena l diseas e i n mal e workers , foun d elevate d risks torsolvents use d a s cleanin g agent s o rdegreaser s (odd s rati o (OR ) 2.5 ; 95 % confidenc e interva l (CI ) 1.56-3.95 ) bu t not fo r exposure t o gasolin e an d diese l fue l (O R 0.98 ; 95 % C I 0.49-1.06 ) o rmoto r an d fue l oi l (O R 1.13 ; 95 % C I 0.69-1.84) . Harringto n e t al . (1989 ) foun d n o associatio n (O R 1.0 ; 95 % C I 0.16-6.3 ) betwee n occupationa l exposur e t o inorgani c solvent s an d glomerulonephritis , bu t th e author s als o conclude d that th e statistica l powe ro l thi s case-referen t stud ywa s no t sufficien t t o detect othe rtha n larg e risk estimates . Th e glomerulonephriti s reporte d i n th e positive epidemiologi c studie s ha s involve d thickenin g o fglomerula rbasemen t membrane s o rdepositio n o fantibodie s against glomerula rbasemen t membrane , a mil d degre e o f abuminuria , an d sometimes tubul e atroph yan d tubula rbasemen t mem bran e thickenin g (Kluw e e t al. , 1984 ; Phillip s e

t al. . 1988) . 3 1

### Othe rindicator s ofrena l functio n hav e als o bee n assesse d i n epidemiologi cstudies

. Levamisole , a dru g use d a s a n antihelminthic , i n cance rchemotherapy , an d i n th e treatmen t o frheumatoi d arthriti s I n humans , fall s int o th e CIGAcategor ybecaus e i t induce s bot h hyalin e drople t an d Os- g accumulatio n i n mal e rat s (Rea d et al. , 1988)

. Base d o n a n absence ofelevate d level s o furinar yNA G i n patient s receivin g 15 0 m g levamisol e pe rdayfo r2 6 weeks , there i s littl e evidenc e t o indicat e that thi s compoun d i s nephrotoxi ci n human s (Diepp e et al. , 1978)

. I n addition , n o positiv e associatio n betwee n urinar yNAG an d acut e o rchroni cexposure wa s note d i n a prevalenc e stud yo f180 dry-cleanin g workers expose d t o tetrachloroethylen e (Sole t an d Robins , 1991)

. Sinc e urinar yNA G i s onl y slightl yelevate d i n mal e rat s expose d t o CIGA , however , urin e chemistr y ma ynot b e a goo d biologica l monito ro fth e typ e o

fnephrotoxicityassociate d wit h CIGA

. I n a stud yo f1 6 female s expose d t o tetrachloroethylen e fro m thei r employmen t i n dry-cleanin g shop s fo ra n averag e o f1 1 years (rang e 1 t o 2 5 years) , Vyskocf l e t al

. (1990 ) foun d n o evidence o frena l damag e excep t fo r a n increas e i n lysozym e i n th e urine

. N o statisticall ysignifican t increas e i n urinar yexcretio n ofP 2 

### -microglobulin , lactat e dehydrogenase , o rglucose , whic h are othe rmarkers o ftubula rdysfunction , were noted

. Th e authors believ e thes e latte rfindings , i n additio n t o th e lac k ofcorrelatio n betwee n intensityo fexposur e an d chang e i n biochemica l parameters , suppor t th e conclusio n tha t rena l damag e i s no t associate d wit h tetrachloroethylen e exposure . Th e evidenc e regardin g rena l injur yi n human s fro m chroni corgani c chemica l exposure i s inadequat e t o demonstrat e whethe ro rno t CIG A exposure ca n affec

t th e huma n rena l tubul e cell

. Existin g report s impl ythat , i fth e associatio n i s real , i t i s th e glomerulu s tha t i s pathologicall yinvolved

. However , thi s ma

ysimpl yreflec

t stud ydesign s whic h concentrate d o n clinica l detectio n o fglomerula reffects

. Sinc e th e injur yt o th e ra t tubul e cell s i s relativel ymild , insensitiv e tests , suc h a s urin e chemistry , whic h are generall yuse d fo revaluatin g human s migh t b e inadequat e t o detec t changes

. D. Factors

Affecting

the Expression

of Alpha u 

### -globulln

Nephropathy Variou s conditions , includin g age , hormon e manipulatio n an d genetics , have th e potentia l fo ralterin g th e expressio n ofCIGA-induce d a^- g nephropathy

. Experimenta l studie s have investigate d th e influenc e o fthes e factors o n CIG Anephrotoxicitya s wel l a s determinin g th e effect s ofa^- g i n femal e rats

. 1. Age-related

effects As discusse d earlier , th e hepati csynthesi s an d urinar yexcretio n o

f 3s- g i n th e mal e ra

t are highl yage-dependent , wit h prepuberta l an d age d animal s showin g negligibl e amounts o fthi s protei n (Neuhau s an d Ftory , 1978 ; Ro yet al. , 1983 ; Richardso n e t al , 1987)

. Accordingly , administratio n o feithe rdecali n t o immature mal e rats (Alde n et al.

. 1984 ) o runleade d

3 2 gasolin e t o aged , 26-mont h old , mal e rat s (Murt yet al. , 1988) faile d produc e rena l cortica l as- g accumulatio n o ra n increas e i n hyalin e drople i 2. Effect of hormone

manipulation A s a^i ) synthesi s i s primaril yunde randrogeni ccontrol , th e effects castration , whic h depresse s hepati csynthesi s o fa_- g (Royan d Neuha i 1967) , wer e explore d b yHobso n e t al

. (1986 ) usin g TMP

. Althoug h significan t increas e i n hyalin e drople t formatio n wa s observe d i n b e castrate d an d uncastrate d mal e F344 rat s expose d t o a singl e ora l dos e TMP , th e severityo fth e lesio n wa s les s i n th e former

. Thus , castrat i diminishe d bu t di d not abolis h th e TMP-induce d nephrotoxicity

. Estroge n i s know n t o inhibi t th e hepati csynthesi s ofa.- g i n th e ra t ( R e t al. , 1975)

. Gar g an d coworkers (1988 ) use d estradio l administratio n stud yth e influenc e ofinhibitio n o fnewsynthesi s ofa^- g o n recover yfr c CIGA-induce d rena l tubul e changes

. Commencin g treatmen t o n th e ni r an d fina l da yofunleade d gasolin e exposure , estradio l reduce d rena l cort h a^- g conten t b y25.41 , an d 5 2 percen t o n post-exposur e days 3,6 , an d respectively , compare d t o rat s receivin g n o hormon e treatment

. A t th e sa r time , hyalin e drople t remova l appeare d t o b e accelerate d i n rat s trea t conjointl ywit h hormones

. Hyalin e drople t numbe ran d siz e (qualitat i observations ) i n hormone-treate d rat s approache d contro l level s a t 3 d a post-exposure , compare d wit h u p t o 9 day s fo rcomplet e resolutio n unleade d gasoline-expose d rat s no t receivin g estradiol

. I n a subsequen t study

. Garg et al

. (1989b ) demonstrate d that pretre men t ofmatur e mal e rat s wit h subcutaneou s injection s o festradio l fo r day s befor e gasolin e exposure completel yinhibite d th e rena l accumulat e o fots- g an a 

### hyalin e droplet s normall yinduce d b ygasoline

. 3. Genetic

variants Th e NB R rat ha s n o detectabl e level s ofhepati cOL- g mRN Ai n eit r sexand , therefore , i s unabl e t o synthesiz e OL- g i n th e live ralthoug h h i constitutiv e level s o fth e mRN Aare presen t i n th e preputia l glan d (Chatter ) et al. , 1989)

. Th e NB R rat i s capabl e o fdevelopin g chemicall yindu e nephropathies , bu t unde rexposur e condition s that produce OL- g nephropa t i n F34 4 rats , d-limonene , TMP , isophorone , an d 1,4-DC B di d not induce a detectabl e OL- g accumulation , hyalin e droplet s o rothe rlesion s i n th e m a NB R rat (Dietric h an d Swenberg , 1991a)

. Identica l result s wer e obtaine d I decali n (Ridde re t al. , 1990 ) an d lindan e (Dietric h an d Swenberg , 19 9 1991a)

. " 4. Alphas-globulin

infusion in female rats Ridde re t al

. (1990 ) intraperitoneal ^ administere d o

_- g (purifie d fr

o mature mal e rat urine ) a

t hourl yinterval s to decali n treatedlemal e Sprag u uawie yrat s fo ra tota l o f8 injection s an d examine d kidne ysample s f nyaiin e droplet s an d o

- - g on e hou rafte rth e las t protei n injectio n ( 9 ho u ane rdecali n treatment)

. Althoug h drople t lormatio n wa s not eviden t mone ysection s fro m th e o^g-infuse d femal e rat s staine d wit h Mallor y

3 3

### Heidenhain , hyalin e droplet an d OL- g accumulatio n were clearl ydemon strate d i n female s expose d t o bot h hydrocarbo n an d mal e urinar yprotein

. B ymean s o ftwo-dimensiona l ge l electrophoresis , th e investigators showe d slight , bu t apparent , rena l cortica l accumulatio n o fots- g i n ,n e 

### ' n,use d 

### females

. Accumulatio n o l th e protei n greatl yincrease d i n female s that wer e bot h infuse d wit h ots- g an d 

### decalin-lreated

. Thes e variou s studie s indicat e a direc t dependenc e o

fCIGA-induce d rena l lesio n expressio n o n th e presence o fOL^-g . E. Chronic

Progressive

Nephropathy Rat s are particularl ypredispose d t o a n age-relate d spontaneou s nephropathy , CPN , that i s mor e severe i n male s tha n i n female s an d tha t affect s certai n strain s mor e tha n others

. CP N i s more commo n i n Sprague Dawleyan d F344 rat s tha n i n th e Wista rstrai n (Gray , 1986) , an d i t i s als o commo n i n th e Osborne-Mende l ra t (Goodma n et al. , 1980)

. Th e etiolog y o fCP N i s not know n bu t th e severit yofth e syndrom e i s influence d b ya numbe ro ffactors , particularl ydietar ymanipulatio n affectin g protei n conten t o rcalori cintake (Masor

o an d Yu , 1989)

. Exacerbate d CPN

. involvin g enhance d severityan d earlie ronse t o fth e disease , i s generall yobserve d afte rchroni cadministratio n o fCIG At o mal e rat s (Trum p et al. , 1984a)

. I t has bee n slate d tha t exacerbate d CP N i s on e componen t (togethe rwit h hyalin e drople t accumulatio n an d granula rcas t formation ) o

fa tria d o flesion s that specifie s th e nephropathi crespons e t o CIG A(Kanerv a et al. , 1987a ; Web b et al.

. 1989)

. Exacerbate d CP N i s usuall yrecognize d alte rmonth s o fcontinuou s treatmen t (Trum p e t al. , 1984a ; Shor

t et al. , 1989a) althoug h Alde n et al

. (1984 ) reporte d earl ysign s afte r2 t o 3 weeks wit h decalin

. Thes e authors (Alde n et al. , 1984) conside r that exacerbate d CP N develop s a s a tertiar yrespons e t o nephro n obstruc tio n cause d byth e CIGA-induce d granula rcasts

. Th e pathologi cfeature s o fCP N (liste d i n Tabl e 4 ) includ e certai n lesion s that are als o foun d i n OL^- g nephropathy , a s wel l a s lesion s tha t ar e distinctive

. Single-cel l necrosis , regeneratin g tubules , an d foca l hyperplasi a o fproxima l tubul e epitheliu m are commo n t o spontaneou s CP N an d t o OL^- g nephropath y(UAREP , 1983)

. CP N i s characterize d bycertai n lesion s that are not components o fo^- g nephropathy , includin g conspicuou s thickenin g o

ftubul e an d glomerula rbasemen t membranes , hyalin e cast s consistin g ofhomogeneous , proleinaceou s materia l (distinc t fro m granula r cast s containin g cellula rdebris) , interstitia l mononuclea rcel l infiltration , fibrosis , tubul e atroph yan d scleroti cglomeruli

. Conversely , earl yan d lat e stage s o fo^ g nephropath yexhibi t a numbe rofcharacteristic s unlike CPN , suc h a s hyalin e droplet accumulatio n associate d wit h ots- g i n tn e P 2 

### segment , granula rcasts a t th e "corticomedullary " junction , an d linea r mineralizatio n i n th e papill a (Trum p et al. , 1984a)

. I n ver yadvance d case s o fspontaneou s CPN , sporadi ctubule s ma ycontai n excessive number s of hyalin e droplet s simila ri n appearance t o thos e induce d b yCIGA

. However , thes e d o not showimmunochemica l evidenc e o fa^- g (unpublishe d obser vations , G.C

. Hard)

. Th e urin e an d seru m chemistr yo fadvance d CP N als o Table 4. Summary

of I* Histopathology

ot Spontaneous

Chronic Progressive ' Nephropathy ot Aging Rats

5 . 6 . 7 . 8 . 9 . Thickenin g o l tubula ran d glomerula rbasemen t membranes . Basophili c segment s ofproxima l tubule s wit h sporadi c mitose s indicative o ftubul e coi l proliferation . Tubula rhyalin e cast s o fproleinaceou s malaria l originatin g i n th e more dista l portio n o l ti e nephron , mainl yi n th e medulla , an d late rpluggin g a considerabl e lengt h o ffee tubule . Foca l interstitia l aggregation s ofmononuclea rinflammator ycell s withi n area s o faffecte d lubules . Glomerula rhyalinizatio n an d sclerosis . Interstitia l fibrosis an d scarring . Tubula ratroph yinvolvin g segment s o l proxima l tubules

. Chronicall yi n advance d cases , occasiona l hyperplasti c loc i i n affecte d tubules . I n som e advance d cases , accumulatio n o fprotei n droplet s i n sporadi c proxima l lubules . 

### differs fro m OL- g nephropathy

. Albuminuria , hypoalbuminemia , an d hypocholesteroremi a typif yCPN , wit h increase s i n seru m creatinin e an d ure a nitroge n level s i n end-stag e diseas e (Barthold , 1979)

. F. Renal

Toxicity Observed

In Chronic Bioassays

of Chemicals

That Induced Kidney Tumors In Rats Fo rth e purpos e ofth e curren t review , bioassay s were identifie d an d dat a were examine d o n seve n chemical s teste d fo rchroni ctoxicityan d carcinogenicit

ybyth e Nationa l Toxicolog yProgra m (NTP ) o rth e Nationa l Cance rInstitut e (NCI)

. Ai l seve n produce d accumulatio n o fhyalin e drop lets , nephropathy , an d kidneytumors i n mal e rats

. Thes e mode l compound s are d-limonene

. dimethy l methylphosphonate

. hexachloroethane , 1,4-DCB , tetrachloroethylene

. pentachloroethane , an d isophorone 3



### . Informatio n wa s als o examine d o n unleade d gasolin e (teste d b yinhalatio n a s a totall y vaporize d for m a

t th e Internationa l Researc h an d Developmen t Corporatio n (IRDC J fo rth e America n Petroleu m Institute)

. Gasolin e i s a comple xblen d wit h CIG Apropertie s (MacFarian d e t al. , 1984)

. Dat a o n th e tw o no n CIGA , Irichloroethylen e andchlorothalonil , are include d lo rcomparativ e purposes . Althoug h extensive acute an d subchroni cstudie s have bee n performe d o n two othe rchemical s (decali n an d TMP)

. bot h o fwhic h caus e ot^- g nephropath y i n th e mal e rat , carcinogenicit ybioassaydat a are not availabl e fo rthes e compounds

.

Severa l o l thes e seve n chemical s canno t b e describe d a s tru e "CIG Acarcinogens " sinc e th e accumulatin g protei n i n th e hyalin e droplet s ha s no t bee n confirme d t o b e a^-g

. Thei rus e a s mode l compound s lo rpurpose s o l developin g th e discussio n o n cance r shoul d no t b e construe d t o mea n tha t al l seve n chemical s ti t th e Polic yStatemen t develope d i n Part I V o l thi s documen t

3 5Trichloroethylene , whic h wa s teste d b y th e NTP , induce s kidneytumors i n mal e rats onl y (NTP , 1988 ) bu t doe s no t caus e a n accumulatio n o fhyalin e droplets o ra n increas e i n ots- g level s (Goldsworth ye t al. , 1988a) . Ther e i s als o som e evidenc e that trichloroethylen e metabolite s bin d covalentl yto rena l macromolecule s (Bruckne re t al. . 1989) . Consequently , thi s com poun d woul d no

t b e considere d a CIGA . Chlorothalonil , a fungicid e teste d o n separat e occasion s by industr yan d a governmen t agency , induce d rena l tubul e tumor s i n mal e an d femal e rat s an d i n mal e mice (NCI , 1978a) . I t als o induce d hyalin e drople t accumulatio n i n proxima l convolute d tubule s o fmal e rats (USEPA , 1988) , bu t thes e may no t becom e apparen t durin g th e firs t fewweeks o

ftreatmen t (Kiltee n e t al. , 1990) . Electro n microscopi c studie s o fmal e rat kidneyfollowin g subchroni c chlorothaloni l exposur e reveale d angula rmembrane-boun d lysosome s containin g crystallin e structures simila rto those observe d i n OL- g nephropath y (writte n communication , Willia m M . Buse yan d Jame s C . Killeen , Experi menta l Patholog yLaboratories , Inc. , t o Offic e o fPesticide s Programs , 1988) . However , oL^- g ha s no

t bee n detecte d i n th e rena l tubule s o f chlorolhalonil-expose d rat s (Swenberg , 1989) . Th e progressio n of chlorothaloni l nephrotoxicityinvolves initially , vacuola rdegeneratio n o f proxima l tubul e epitheli u m followe d 4 weeks late rby tubul e cel l hypertrophy , hyperplasia , an d tubul e dilatio n (Killee n et al. , 1990) . Therefore , thi s compoun d appear s not t o produc e th e sam e spectnj m o rsequenc e o f lesion s induce d b

yCIGA . Furthermore , chlorothaloni l ha s bee n show n t o interact wit h cellula rmacromolecule s includin g historie s an d thio l proteins , possibl ythroug h covalen t bindin g o fa metabolit e wit h sulfhydry l group s (Rosanof fan d Siegel , 1981) . Chlorothaloni l als o induce s overt rena l dysfunctio n i n bot h sexe s o frats . A t dose s fro m 4 0 mg/kg/day , bloo d ure a nitroge n an d creatinin e wer e increase d whil e circulatin g level s o fglucos e an d albumi n wer e decrease d (USEPA , 1988) . Fo rthes e variou s reasons , chlorothaloni l i s no

t considere d a membe ro fth e CIG A class . A summar

yo fth e nonneoplasti c an d preneoplasti c kidne yeffect s observe d i n mal e rat s afte radministratio n o fth e eigh t substance s selecte d a s possibl e CIGA i s presente d i n Tabl e 5 . Non-neoplasti c an d preneoplasti c lesion s reporte d i n femal e rat s an d mic e o fbot h sexe s ar e summarize d i n Tabl e 6 . Th e dat a i n thes e tw o Table s wer e extracte d fro m th e NT P Technica l Report s (se e Tabl e A- 2 i n th e Appendix) an d othe rrelevan t literature . I n mal e rats , at leas t on e cas e o frena l tubul e cel l hyperplasi a wa s reporte d i n th e 2-yea rbioassay s lo rth e seve n rena l carcinogen s teste d by th e NTP . Th e incidenc e wa s generall ymuc h highe ran d dos e responsive . Althoug h no

t reporte d i n th e IRD C bioassay , thi s lesio n wa s observe d i n late r researc h studie s o

funleade d gasolin e (Shor t e t al. , 1989b) . Non e o

fth e eigh t bioassaye d substance s produce d tubul e cel l hyperplasi a i n femal e rats , althoug h thi s lesio n wa s reporte d i n mal e mic e expose d t o tetrachloroethylene . I n mal e rats , rena l change s describe d a s "toxi c tubula r nephropathy " (encompassin g degeneratio n o ftubul e epithelium , necrosis , 

# I

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Table 6. Summary

of Dam from 2-year Bioassays

on Non-Neoplastic

and Pre-NeepJastic

Kidney Lesions in Mice and Female Rats Exposed

to Eight Model Compounds

that Induced Renal Tumors in Male Rats

Chemica l Hyalin e Droplet s Toxi c Nephropath y Cas t

Formatio n Mineralizatio n Karyomegal y Hyperplasi a 1,4-Dichlorobenzen e Dimethy l methyl phosphonat e Hexachloroethan e Isophoron e d-limonen e Pentachloroethan e Tetrachloroethylen e

Unleade d Gasolin e -

- "

-

-

- "

- +

(femal e rat s an d mal e mice )

-

+ (femal e rat s an d mice )

-

-

- N R

- -

-

+ (mic e hyaline )

-

-

- + (mice )

- +

(femal e rats p

-

+ mic e (C a deposition )

-

-

- '

- N R

N R N R

N R N R N R (femal e rat s an d mice)* 5

+ -

- *

-

-

- • (mal e mice) 0

- + Positiv e Negativ e N P No t reporte d C a Calciu m Incidenc e an d severit ymuc h highe ri n mal e rat s Highes t incidence s i n mal e ra t



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## Par t 2 . Carcinogenicit y

Th e secon d majo rpar

t o fthi s documen t compare s an d contrast s informatio n o n kidne ytumors induce d b yclassica l rena l carcinogen s wit h informatio n fro m th e NT P (o rNCI ) assay s fo rrena l neoplasi a induce d b y chemical s that produce d hyalin e droplet s and/o raccumulatio n o fots-g

. , n 

### addition , othe rinformation , suc h a s mutageni cactivityan d tumor-promotin g ability , whic h hel p t o defin e a CIG Acarcinoge n o rpoin t t o possibl e mecha nis m ofaction , are evaluated

. Epidemiologica l studie s ofhuma n rena l cel l cance rare reviewe d fo r consistencywit h th e hypothesi s that CIGA-induce d rena l cance ri n mal e rat s i s a n inappropriat e endpoin t fo rassessin g huma n risk

. Implici t i n thi s evaluatio n i s a presumptio n ofmal e rat-to-huma n tumo rsit e concordance , a suppositio n EP

Agenerall ydoe s not make

. I n thi s specia l case , however , th e hypothesize d mechanis m bein g examine d depend s o n th e accumula tio n o ftow-molecular-weigh t protei n i n th e rena l tubule , regardles s o f species

. Hence , th e predicte d targe t sit e fo rcance ri n humans , a s i n th e rats , woul d b e th e rena l tubule

. V . Pathologi c Feature s ofRena l Carcinogenesi s Induce d byClassica l Carcinogen s Amon g th e man ychemical s recognize d a s inducers o froden t cancer , severa l have bee n use d a s mode l kidne ycarcinogen s fo rstudyin g th e pathogenesi s ofrena l tubul e tumors i n rats

. Thes e ar e dimethylnitrosamin e (DMN) , diethylnitrosamin e (DEN) , N-nitrosomorpholine , N-elhyl-N hydroxyethylnitrosamin e (EHEN) , lea d acetate , N-(4-fluoro-4 - biphenylyl)acetamid e (FBPA)

. an d anatoxi n B , (Hard

. 1990)

. I n th e mouse , certai n nitrosamines , streptozotocin

. an d ochratoxi n Aare stron g inducers o frena l tubul e tumors , whil e th e classica l rena l carcinoge n i n hamsters i s diethylstilbestro l (Hard

. 1987)

. I n general , thes e prototypi crena l carcino gen s ar e activ e i n bot h male s an d females

. Studie s o n th e pathogenesi s ofrena l tubul e tumo rformatio n usin g mode l carcinogen s i n ral s demonstrat e tha t a continuu m o l chemicall y induce d step s lead s Iro m atypica l hyperplasi a i n tubule s (als o terme d hyperplasti ctubules , tubul e dysplasia , an d atypica l cel l foci ) throug h micro scopi cadenomas , t o macroscopi cadenocarcinoma s o rcarcinoma s (Hard , 1987 ; Lipsk

yan d Trump , 1988)

. I n addition , there are invariabl ypathologi cchange s whic h preced e th e proliferativ e sequenc e o fpreneoplasti can d neoplasti clesion s includin g a

4 1period o

fearl ynephrotoxicit

yand . often , karyomegaly . Thes e variou s lesion s are describe d belo wi n chronologica l sequence . A. Early Nephrotoxicity Acut e toxi c change s occu ri n th e proxima l tubule s shortl yafte rth e administratio n o

fclassica l rena l carcinogens . The yinclud e mil d lipi d drople t accumulatio n an d scattere d single-cel l necrosi s (Hard , 1987) . Dependin g on (h e carcinoge n used , thi s earl y damag e ca n b e observe d i n differen t segment s o fth e rena l tubule . Fo rinstance , wit h DM N i t i s localize d to th e P2 segmen t (Har d et al. , 1984 ) an d wit h FBPA , t o th e P 3 segmen t (Dee s e t al , 1980a.b) . Detaile d histologica l and/o rultrastructura l observatio n show s tha t hya line drople

t accumulatio n i s no

t induce d b

yDM N (Har d an d Butler , 1971 ; Har d e t al. . 1984 ) o rDE N (G.C . Hard , unpublishe d observations) ; no rha s I I bee n describe d i n studie s usin g othe rcarcinogens , suc h a s FBP A (Dee s e t al. . 1980a,b) , a s model s lo rrena l carcinogenesis . More i s know n abou t DM N tha n othe rclassica l rena l carcinogen s concernin g molecula rinteraction s durin g th e tim e tha t acut e toxi c change s ar e see n i n th e proxima l tubules . DN A adduct formatio n i n rat rena l tissu e occur s rapidl yfollowin g a singl e administratio n o fDMN . 0*-Methylguanin e forme d i n th e rena l corte

x(Fa n e t al , 1989 ) persist s a

t leas t 4 day s postinjectio n (Nicol l e

t al , 1975) , whic h i s consisten t wit h th e notio n tha t methylatio n o

fth e 0 * positio n o

fguanin e i n DN A i s th e most likel yinitiatin g even t (Pegg , 1984) . 0 . Karyomegaly Conspicuou s nuclea renlargement , indicative o fincrease d chromosom e numbe rwithou t completio n o l mitosi s (Jackson , 1974) , mayoccu ri n scattered proxima l tubul e cell s durin g th e week s precedin g developmen t o f carcinogen-induce d proliferativ e foci . Althoug h karyomegal yi s produce d b y many, but perhap s no

t al l rena l carcinogens , ther e i s n o evidenc e tha t thes e cell s participate i n th e initia l formatio n o fproliferativ

e foci . Henc e karyomegal y i s no t regarde d a s a preneoplasti c lesio n (Dee s et al , 1980a ; Hard , 1987 ; Lipsk yan d Trump . 1988) . C . Tubule

Cell Hyperplasia Tubul e cel l hyperplasi a lead s to th e appearanc e o ftubule s wit h prolif

eratin g epithelium , usuall ymult Havered , that partiall yo rcompletel yfill s th e tubula rlumen . Althoug h lumena l dilatio n ma yb e pronounce d (sometime s t o cysti c proportions) , th e structur e o fth e individua l tubul e remain s intac t with a confluen t basa l lamina . Affecte d cell s ma yb e eosinophilic , baso philic , o rpale-stainin g an d ofte n wit h vesicula rnucle i an d prominen t nucleoli . Mitoti c figure s are variable . A s a preneoplasti c lesion , th e hyperplasti c tubul e i s usuall yassociate d wit h som e degre e o fcellula ratypi a (dysplasia) i n th e for m o fcel l pleomorphis m an d increase d nuclea rto cytoplasmi c are a rati o (Hard , l987;LipskyandTrump . 1988) . Preneoplasti c tubule hyperplasi a i s generall yconsidere d to b e distinguishabl e fro m th e backgroun d tubula rregeneratio n that i s a componen t o fspontaneou s CP N (Lipsk yan d Trump , 1988 ; NTP . 1988) . O . Adenoma Adenoma s ar e smal l neoplasti c foc i representin g epithelia l cel l proliferatio n beyon d th e well-define d structure o findividua l tubules . Thes e lesion s ar e soli d o rcysti c i n for m an d th e cellula rmorpholog yan d architec tura l appearanc e i s simila rto tha t o fadenocarcinomas , whic h ar e describe d below , particularl y th e well-differentiate d variants . Wherea s adenoma s an d hyperplasti c tubule s ca n b e differentiate d o n th e basi s o ffinit e structure , th e distinctio n betwee n adenoma s an d adenocarcinomas/carcinoma s i s a n arbitrar yon e base d primaril yo n size . Neoplasm s i n th e ra t kidne yparen chym a les s tha n approximatel y0. 5 c m ten d to lac k significan t vasculariza tion , hemorrhage , an d degeneration , althoug h ther e ma yb e single-cel l necrosis , mitosis , an d cel l pleomorphis m (Hard , 1990) . E. Adenocarcinomas and Carcinomas Rena l tubul e tumor s compris e histologica l variant s base d o n stainin g characteristic s an d architectura l organization . I n th e rat , rena l tubul e tumor s consist mainl yo

flightl ybasophilic , granular , and/o rclea rcell s organize d i n tubular , lobular , solid , o rpapillar ypatterns . Glandula rdifferentiatio n a s oppose d t

o soli d sheet s o

fcell s distinguishe s adenocarcinoma s fro m carcinoma s bu t an yclea rdistinctio n betwee n adenocarcinoma s an d carci noma s i s ofte n meaningles s becaus e o l admixture o l bot h well-differenti ate d an d poorl ydifferentiate d area s withi n th e sam e tumor . Increase d cellula rpleomorphis m tend s t o correlat e wit h a decreasin g degre e o ftubula r differentiatio n an d anaplasti c variant s occu roccasionally . Cell s withi n adenocarcinoma s maintai n man yo fth e ligh t an d electro n microscopi c characteristic s o l proxima l tubul e epithelium , i n particular , microvill i resemblin g brus h border , basemen t membrane , an d cytoplasmi c vesicles . Brus h borde rmayoccu rinappropriatel ybetwee n adjacen t cells , alon g an ycel l border , o ra s intracellula rprofiles . Adenocarcinomas/ carcinoma s ar e wel l vascularize d an d usuall ydisplayarea s ofhemorrhag e an d degeneratio n (UAREP , 1983 ; Lipsk yan d Trump , 1988 ; Hard . 1990) . F . Tumor

Progression Rena l tubul e tumor s o

fth e ra t are slowl ygrowin g neoplasm s usuall y requirin g abou t 4 0 week s t o becom e clinicall ypalpabl e i n most experimenta l system s (Hard , 1987) . The ycan gro wt o larg e dimensions , severa l centimeters i n diameter . Unlik e thei rspontaneousl yoccurrin g huma n counterparts , rena l tubul e tumors induce d i n rat s bychemica l carcinogen s metastasiz e infrequentl y (Lipsk yan d Trump , 1988) . However , lifespa n o fth e anima l i n chronic exposure regimen s mayb e a limitin g factor . Single-dos e studie s wit h DMN , whic h maximize th e life-spa n followin g tumo rinitiation , have demonstrate d a lin k betwee n surviva l period , tumo rsize , an d incidenc e o fmetastasi s i n rena l carcinogenesi s (Hard , 1984) . Fo rexample , rat s tha t survive d a

t leas t 1. 5 year s afte rdosin g wit h DM N showe d a hig h rat e o fmetastasis ,

Al

### approximatel y5 0 percent , wheneve repithelia l tumo rdimension s exceede d

2. 4 cm . Thes e dat a confir m th e malignan t potentia l o frena l tubul e tumor

s induce d i n th e ra

t bya classica l carcinogen

. G. Site of Origin of Renal Tubule Tumors The precis e locatio n withi n th e nephro n fro m whic h experimenta l rena l tubul e tumors aris e varie s wit h th e carcinogen , an d correlate s wit h th e sit e o fth e induce d earl ynephrotoxicity

. Thus , th e P 3 segmen t i s th e sit e oforigi n fo rFBPA-induce d tumors (Dee s et al , 1980a.b) , whil e DM N tumors aris e fro m th e convolute d segment s o fproxima l tubules , probabl yP2 (Hard , 1990)

. Lead-acetat e an d DEN-induce d tumors appea rt o originat e i n bot h P2 an d P 3 segment s (Nogueira , 1987)

. VI. Neoplastic

and Preneoplastic

Lesions

Observed

In the 2-Year Bioassays Data fo ral l reporte d rena l tubul e tumor s an d tubul e hyperplasi a i n mal e rats fro m th e 2-yea rbioassays o n th e eigh t mode l substance s are summa rized i n Tabl e 7

. Fo rtumors occurrin g a t site s othe rtha n th e rena l tubule , onl ystatisticall ysignifican t incidence s are mentioned

. Tabl e 8 provide s simila rinformatio n fo ririchloroethylen e an d chlorothalonil

. Fo reigh t ofth e ten substances , exposure wa s b yth e ora l route ; fo rtwo , i t wa s b yinhalation

. Th e experiment s wer e conducte d ove rapproximatel ya decade , whic h ma y accoun t fo rth e lac k

ofstandardize d terminolog yi n describin g lesion s an d difference s i n attentio n pai d t o th e possibl e rol e ofchemicall yinduce d ots- g accumulatio n i n th e mal e ra t kidney

. I n a separat e se t ofanima l bioassays conducte d fo rth e military , mal e rats were expose d 6 hours/day , 5 days/Wee k fo r1 yea rt o th e syntheti c hydrocarbo n missil e fuels , RJ- 5 an d JP-10

. A t termina l sacrific e afte r2 years , thes e animal s ha d evidence o fnephropath ycharacteristi co fot^- g accumulatio n an d significan t increase s i n rena l tubul e tumors ( 9 i n 6 5 animal s expose d t o RJ- 5 a t 15 0 mg/m 3 

### ; 9 i n 5 0 animal s expose d t o JP-1 0 a t 56 2 mg/m 3

### ) (Bruner , 1984 ; MacNaughto n an d Uddin , 1984)

. I n contrast , th e kidneys o

ffemal e rat s an d femal e C57/BL 6 mice similarl yexpose d t o RJ 5 o rJP-1 0 wer e unaffected

. Likewise , non e o fth e animals , includin g mal e rats , expose d t o RJ- 5 continuousl yfo r9 0 day s an d hel d 1 9 additiona l month s before sacrific e develope d rena l tubul e tumors

. I n additio n t o th e specifi cresults obtaine d fro m individua l bioassays , there are consideration s generi ct o al l bioassays conducte d b yth e NTP

. Fo r example , th e NT P position s wit h regar d t o evaluatio n o

frare tumors an d th e us e o fhistorica l control s influence NT P interpretatio n ofth e evidenc e fo r carcinogenicityo fCIG A(Hasema n et al , 1984)

. Likewise , surviva l rate s influenc e th e abilit yt o analyz e informatio n fro m anima l bioassays

. Thes e generi cissue s ar e explore d befor e describin g th e result s o findividua l studies

. A. Generic

Considerations Rena l tubul e tumors are neoplasm s wit h a lo wbackgroun d incidenc e i n laborator

yanimal s includin g th e ra t strain s use d i n th e chroni cbioassays 4 4 Table 7. Chemica l Incidences of Renal Tubule Preneoplasia

and Neoplasia

in Rats taken from i year Bioassays

on Eight Model Substances Strai n Se x 1,4-Oichloro benzen e (NTP-TR-319 ) (NTP . 1967a ) Gavag e F34 4 Change Dose * (mg/kg/day ) 0 15 0 30 0 M Surviva l (% ) 7 7 6 9 4 3

Hyperplasi a (% ) 0 2 1 8 Adenoma s Incidenc e 0/5 0 0/5 0 1/5 0 Adj . Rat e (% ) 0 0 4 Adenocarcinoma s Incidenc e 1/5 0 3/5 0 7/5 0 Adj . Rat e (% ) 3 9 2 6 Comptrtfl d Incidenc e 1/5 0 3/5 0 8/5 0 Adj . Rat e (% ) 3 9 2 8 Othe rTumors : HepatocePuta rtumor s i n mice . Chemica l Strai n Se x Chang s Dimethy l methyl phosphonat e (NTP-TR-323 ) (NTP . 1987b ) Gavag e F34 4 M Surviva l (% ) Hyperplasi a (% ) Adenoma s Do s

0 5 6 0 i (mg/kg/day ) 50 0 100 0 3 4 1 6 Non e 1 9

1 8

Admocararrama s Incidenc e 0/5 0 2/5 0 3/4 9 Ad j Rate (% ) 0 9 1 9 Othe rTumors : Mononuclea rcel l leukemia ; transitiona l cel l papilloma s ofth e rena l pelvis .

Chemica l Strai n Se xChange s Dose s (mg/kg/day ) 0 0 21 2 42 3 Hexachloro ethan e (NTP-TR-68 ) (NCI . 1978b ) Gavag e Osborne Mende l M Surviva l (% ) 5 6 Hyperplasi a (%) 6 5 2 0 1 8 No t Reporte d Adenoma s Incidenc e Adj . Rate 0/2 0

0 0/1 8 0 4/3 7 1 1 0/2 9 0 Carcinom a Non e Othe rTumors : Hepatocellula rtumor s i n mice .

(cont. )

4 5Table

7. (cor I) Chemica l Strai n HexactHoroethan e (NTPTR-361 ) (NTP , 1989 ) Gavag e

Othe rTumors : Chemica l Isophoron e (NTP-TR-291 ) (NTP . 1986a ) Gavag e

OAfl L Tumor s Chemica l d-Limonen e (NTP-TR-347 ) (NTP . 1990 ) Gavag e

Othe rTumor s rj4 4 Sex M Change s

Surviva l (% ) Hyperplasi a (% ) Adenoma s Incidenc e Adj . Rate (% ) Incidenc e Adj . Rat e (% ) Comhiimd , Incidenc e Adj . Rat e (% ) Dose s (mg/kg/day ) 0 1 0 2 0 6 2 4 1/5 0 3 0/5 0 0 1/5 0 3 5 8

8 2/5 0 6 0/5 0 0 2/5 0 6 5 2 2 2 4/5 0 1 5 3/5 0 9 7/5 0 2 4 Margina l increas e i n pheochromocytoma s i n mal e rate 3*. m SexChange , Doae . (mg^/day ) 0 t M Mi l F34 4

Preputia l gla r tumors , an d Strai n F34 4 M Surviva l (% ) Hyperplasi a (% ) Adenoma s Incidenc e Adj . Rate (% ) Adenocarcinoma s Incidenc e Adj . Rat e (% ) Combine d Incidenc e Adj . Rat e (% ) t d tumors i n mal e rats ; hepatocetlut i malignan t lymphoma s i n mal e mic e SexChange * M

Non e i n mice o rrat s 4 6 Surviva l (% ) Hyperplasi a (% ) Adenoma s Incidenc e Adj . Rat e (% ) Adenocarcinoma s Incidenc e Adj . Rat e (% ) Combine ^ Incidenc e

_ A d i.B?!ei%l_ . 6 6 0

0/5 0 0 0/5 0 0 0/5 0 0 6 6

2

0/5 0 0 3/5 0 9 3/5 0 9 2 8

8

2/5 0 8 1/5 0 4 3/5 0 1 2 i rtumors , mesenchyma l Dose s (mg/kg/day ) 0 7 S IS A 6 0 0

0/5 0 0 0/5 0 0 0/5 0 0 6 8

4

4/5 0 1 2 4/5 0 1 2 8/5 0 2 3 6 9

7

8/5 0 1 9 t 3/5 0 ! 7 

### 1

1 11/5 0 , 2 5

i Table 7. (cont.i Chemica l Pentachloro ethan e (NTP-TR-232 ) (NTP . 1983 ) Gavag e

I

1 Othe rTumors :

Chemica l i

' Tefrachtoro ethyfen e (NTP-TR-311 ) (NTP . 1986b ) Inhalatio n

OthflfTumora : Mixtur e Unleade d gasolin e (USEPA , 1987 ) Inhalatio n

Othe rTumors : Strai n

F34 4

Hepatocellula i Strai n F34 4 Se x

M Change *

Surviva l (%) Hyperplasi a (% ) Adenoma s Incidenc e Adj . Rate (% ) Incidenc e Adj . Rate (% ) Combine d Incidenc e Adj . Rate (% )

•tumors i n mice . Se xChange s M Surviva l (%)

Hyperplasi a (% ) Adanfltna a Incidenc e Adj . Rat e (% ) Incidenc e Adj . Rat e (% ) Combine d Incidenc e Adj . Rat e (% ) Leukemi a i n rats ; hepatocellula rtumors i n mice . Strai n Se xChange s 0 F34 4 M Surviva l (% ) Hyperplasi a (% ) Adenoma s Incidenc e 0/4 9 Adj . Rat e (% ) 0 Carcinoma

s Incidenc e 0/4 9 Adj . Rate (% ) 0 Combine d Incidenc e 0/4 9 Adj . Rat e (% ) 0 Hepatocellula rtumor

s i n femal e mice .

4 7 Do** * 0 8 2 0 075 0 0 1/5 0 2 1/5 0 2 i (mg/kg/day ) 7 5 6 8 0 1/4 9 3 1/4 9 3 2/4 9 6 ia w 5 2 2 475 0 1 4 0/5 0 0 4/5 0 1 4 Doaee(ppm ) 0 20 0 40 0 4 8 0 1/4 9 4 0/4 9 0 1/4 9 4 4 0

6 3/4 9 1 1 0/4 9 0 3/4 9 1 1 2 4 1 0 2/5 0 1 1 2/5 0 1 1 475 0 2 2 Doaee(ppm ) 6 7 29 2 20S 8 No t affecte d Not reporte d i n th e bioassa y 1/5 9 2/5 6 1/4 5 2 4 2 1/5 9 2/5 6 6/4 5 2 4 1 4 1/5 9 5/5 6 7/4 5 2 9 1 6Tables.

Chemloa l Incidences cl Renal Tubule Preneoplasia

end Neci*asia

in Rats taken *w year Bioassays

on Chlorothaloni

and Trichktroethytone Strai n Se x Change * (PP»n ) SOW1012 6 CMorotia lofn l (NTPTR-41 ) (NCI . 1978a )

Die t Osbome - M Surviva l (% ) Hyperplasi a (% ) Incidenc e Rate(% ) 

### Qatdnama* Incidenc e Rate(% ) ComMne d Incidenc e Rale(% ) Surviva l (% ) Hyperplasi a (% ) 

### Adflnomn g Incidenc e Rata(% ) 

### Cardnoma a Incidenc e Rate(% ) 

### Combinfl d 8 2

0/1 0 0 4 0

2/4 6 4 4 0

1/4 9 2 0/1 0 1/46 3/4 9 0 2 6 0/1 0 0 5 0

0/1 0 0 0/1 0 0 3/4 6 6 6 2

0/4 8 0 1/4 8 2 4/4 9 8 7 2

3/5 0 6 2/5 0 4

OjhfltlurflflC S Chemica l non e

Strai n Se x Incidenc e Ra»(% )

Change * 0 0/1 0 1/4 8 5/5 0 0 2 1 0

Do** * (mg/kg/day ) 0 90 0 100 0 Trichloro ethylen e (NTP-TR-273 ) (NTP . 1988 ) Gavag e Osbome - M Surviva l (% ) Hyperplasi a (% ) Adenoma s Incidenc e Adj . Rat e (% ) Carcinoma s Incidenc e Adj . Rat e (% ) Cflmbjifl d Incidenc e Adj . Rat e (% ) 4 2

0 0/5 0 0 0/5 0 0 0/5 0 0 4 4 0 0/5 0 0 0/5 0 0 0/5 0 0 3 4 1 0 6/5 0 3 2 0/5 0 0 6/5 0 3 2 3 0 6 1/5 0 6 1/5 0 6 2/5 0 1 1 Tumor s i n Othe rStrains : 2-4 % rena l tubul e tumors i n thre e othe rstrains . Oma rTumors : Malignan t mesotheliom a i n mal e rats : hepatocellula rtumors i n mal e an d

femal e mice , an d lymphom a i n femal e mice .

4 8 

### o n CIGA , namel y F34 4 an d Osbome-Mendel . TMJ*«#"gZ£** dance o fthese tumor

s m mal e F34 4 rats i s <» n 

### » ^ r 

### ^JS t 

### ?^.° u 

### S 2 percent based o n data reported o n 1,943 animal s whic h served as vjht o control s I n studiesinvolvin g administratio n o fchemical s vi a oorri10Iigava c (NTP . 1990) . I n a large rhistorica l contro l database . Involving^.32 0 m a 

### an d 2.37 0 femal e F34 4 rats used a s untreated control s I n NTPtwo-ye i bioassaya . th e Incidenc

e was 0.3 5 percen t fo rmale s an d 0.1 7 pwcenti M females suggestin g a mal e predilectio n fo r« n 

### a ,tu 

### ^ t e 

### J u 

### ^ 1 l 

### ?iSS2 S al. . 1984) . Thi s I s supporte d byspontaneou s rena l »"^ * * TM ,I 

### £ * E rate s recorde

d forOsborne-Mende l rats used a^ntrol s I * .th a N < Carcinogenesi s Testin g Progra m (Goodma n e t al. . l^L^ 9 7 

### *TM"** * 97 0 females , the Incidenc e wa s 0. 3 percent an d 0 i*«*nt . r 

### «*»25 S Becaus e o fth e Infrequenc

y o frena l tubul e tumors , eve n margina l mcraa w I n thei rIncidenc

e m treate d animal s (statistical!) f••onWIcan

t ^norjmpar i t o historica l rathe rtha n concurren t controls

) *W*^}%JTMTMr\ lototoojlcaliysignlficartandattrl) ^ etal . 1984 ; NTP . 1989) . I n th e 2-yea rstudie

s wit h th e eight selected rena l carcinogens , t l observe d Incidence

s o frena l tumor

s fo rIndividua l chemically dosed grou t wer e less than2 5 percent , an d n o hlghertha n 16pe^'°rmc-s t Becauj o fth e lo wbackgroun d rat

e I n bot h concurrent an d historica l contro l however, developmen

t o frena l tubul e tumor

s a t thes e Incidence

s w t ascribe d to a n effec

t o fth e chemical . Th e NT P bioassay

s provid e littl e Insigh

t Int o th e histogenesi s oft ] rena l tumor

s a s the y wer e designed an d performe d wit h the

i prim e objec j o fdeterminin g th e presence o rabsenc e o fcarcinogeni c activit

y o fth e a chemical . Althoug h a n Industry-sponsore d stud y o funleaded gaao P include d Interi m sacrifices , eve n thi s bioassa y di d no t ,n 

### corporate sei sacrifice s designe

d t o provid e informatio n o n th e sit e o forigi n o rhlstoge esi s o ftumors . J Surviva l rate

s I n high-dose mal e rat s wer e poo ri n severa l o

fth e N T bioassays . whic h complicate s Interpretatio n o

fth e data^h«hlohn»rtal i rat e observe

d I n some o

fthese studie

s canno t be attributed to th e w j tumor s (Hoe l e t al. . 1988) . I n fact , poo rsurviva l rates usuallyInoTca U excessiv e toxicity. Fo rth e 1.4-DC B bioassay . surviva l o fthenhtyvd M male s 4 0 percen

t at termination , becam e significantly lowe rtha n tha n vetitet e control s afte rwee k

9 7 (NTP . 1987a) . Neart y al l death s we

j nonacclderital . A simila rsituatio n pertain s t o isophorone wher e onl y a percent o fhig h dos

e males survive

d to terminatio n (NTP . 1986a) . I Th e decrease

d surviva l rates sugges

t tha t a maximu m tolerated dot . (MTO) was exceeded sinc e th e earl y death s coul d no t b e attribute

d I tumors . Administratio n o fa chemica l a

t dosesexceedjnga n MTDmay a m response s tha t woul d b e see n a

t lowe rdose level s (Offic e o fScienc e a n Technolog

y Polic y (OSTP) . 1985) . However , exceedingja n MTD, tyn m i s no t compellin g evidenc

e tha t tumor s are produce

d onlyrwhe n d etoxfflc i tio n mechanisms ar

e overwhelmed . I n fact , survlvalo fmal e rat s n tow-dos groups administered isophorone . 1.4-DCB . hexachtoroethan e an tetrachloroethane wa

s equivalent t

o tha t o fth e concurren

t contro l grou p 4 9 toan d rena l tumo rincidenc e wa s elevate d i n thes e animals . Surviva l wa s

excellen t fo ral l dos e group s o fmal e rat s administere d d-limonen e o r unleade d gasoline . However , it i s difficul t to compare tumo rincidence s amon g studie s wit h marke d difference s i n surviva l rates , especiall ywhe n ther e i s th e potentia l fo rdevelopmen t o fslow-growin g tumors , suc h a s rena l neoplasms . B. Renal

Tumor Incidence Amon g th e eigh t mode l carcinogens , th e overal l unadjuste d incidenc e rate s fo rrena l tubul e tumors (adenoma s an d adenocarcinomas/carcinoma s combined ) i n mal e rat s range d fro m 3 percen t t o 1 1 percen t at low-dos e level s an d 0 percen t t o 2 2 percen t at th e hig h dose . Th e highest unadjuste d incidenc e (22% ) wa s associate d wit h d-limonene . Fo rth e remainde ro fth e chemicals , incidence s o frena l tumor s wer e 1 6 percen t o rless . Whe n adjuste d fo rintercurren t mortality , th e incidenc e rate s fo rcombine d rena l tumors range d fro m 0 percen t t o 2 8 percen t wit h 1,4-DC B bein g th e highest (Tabl e 7) . Fo ral l o fth e eigh t mode l carcinogens , an d als o fo rtrichloroethylen e an d chlorothalonil , th e increas e i n th e incidence s o frena l tubul e tumors , wher e adjuste d fo rintercurren t mortality , wa s dos e related . Becaus e th e incidenc e o frena l tubul e tumors wa s lo wan d ther e wer e confoundin g factors suc h a s toxicit yoccurrin g a

t al l dos e level s i n most studies , i t i s no t possible , fro m th e NT P bioassaydata , t o determin e i fther e wa s a relationshi p betwee n increasin g dos e an d percentag e o ftumors classifie d a s adenocarcinoma s rathe rtha n adenomas . I n th e 198 6 Guideline s fo rCarcinoge n Ris k Assessment , EP A discusse d its strateg yfo ranalyzin g combination s o f benig n an d malignan t tumors (USEPA , 1986) . I n general , th e Agencystate d tha t i t woul d conside rth e combinatio n o fbenig n an d malignan t tumors t o b e scientificall ydefensibl e i fth e benig n tumor s have th e potentia l t o progres s to th e associate d malignancie s o fth e sam e histogeni c origin . Th e weight of-evidenc e tha

t a chemica l i s potentiall ycarcinogeni c fo rhuman s woul d increas e whe n ther e i s a dose-relate d increas e i n th e proportio n o ftumor s tha t are malignant . Conversely , i fonl ybenig n tumors wer e observed , thi s woul d constitute les s evidenc e o fhuma n cance rpotential . Sinc e th e distinctio n betwee n adenoma s an d adenocarcinoma s fo rrena l tubul e tumor s i n rats i s rathe rarbitrary , base d mainl yo n size , thes e genera l principles canno

t b e rigidl yapplied . C. Histogenesis

of Renal Tumors A s previousl yindicated , NT P bioassay s ar e designe d t o determin e whethe ro rno

t a chemica l i s a carcinogen . The yare no t designe d wit h th e inten t o fprovidin g informatio n to evaluat e th e developmenta l stage s o frena l neoplasia . Althoug h rena l tubul e hyperplasi a wa s reporte d i n th e mal e rat fo rseve n o l th e eigh t bioassays an d incidence s o fthi s lesio n generall y increase d wit h increasin g dose , furthe rinsigh t wit h respec t t o histogenesi s int o possibl e interrelationship s betwee n hyperplasia , adenomas , an d carci nomas i s no

t possibl e becaus e o

fth e lo woveral l frequency o

fthes e lesions . Th e occurrence togethe ro fpreneoplasti c an d neoplasti c lesion s i n most

5 0 studie s wit h th e eigh t chemical s doe s provid e indirect evidenc e o fprogres sio n fro m tubul e cel l hyperplasi a vi a adenoma s t o adenocarcinomas . I n studie s wit h d-limonen e (NTP , 1990 ) an d hexachloroelhan e (NTP , 1989) . thes e lesion s wer e state d t o b e par t o l a continuou s morphologi c spectrum . Thi s accord s wit h th e generall yaccepte d viewo n rena l tubul e tumo r formatio n an d progressio n (Lipsk yan d Trump . 1988 ; Hard . 1990) . D. Renal

Tumor Latency

and Progression Rena l tubul e tumors produce d b yadministratio n o

fCIG A appea rt o b e late developin g neoplasms . Time s a t whic h suc h tumors wer e firs t observe d i n bioassay s o

fth e eigh t mode l carcinogen s usuall yexceede d 1 8 months . I n general , th e firs

t rena l tumo robserve d i n eac h o fth e bioassay s occurre d abou t 5 t o 1 0 week s earlie ri n th e high-dos e tha n i n low-dos e animals . Becaus e rena l tubul e tumors ar e no t immediatel ylife-threatening , the ywer e usuall ydetecte d i n bioassays a t termina l sacrific e o rat deat h o fth e anima l fro m othe rcauses . Ou t o fth e eigh t bioassays , ther e wa s onl yon e cas e o f rena l tumo rmetastasis , occurrin g i n th e high-dos e grou p o l hexachloroeth an e (NTP . 1989) . E. Induction

of Other Tumor Types Si xo fth e eigh t mode l substance s produoe d live rtumor s i n mal e and/o r femal e mic e bu t no

t i n mal e o rfemal e rats . Thes e chemicat e wer e hexachloroelhane , unleade d gasoline , isophorone . 1,4-DCB . pentachloroethane , an d tetrachloroethylene . A difieren t mechanism , inde penden t o

fhyalin e droplet accumulation , ma yb e involve d i n th e productio n o flive rtumor s b

y thes e si xchemicals . Som e author s suggest a mechanis m involvin g peroxisom e proliferatio n t o accoun t fo rth e productio n o fsuc h live r tumors (Elcomb e e t al. , 1985 ; Goldsworth yan d Popp , 1987) . A n alternative explanatio n to rth e live rtumor s i s that bot h CIGA-induce d live ran d kidneytumors ar e produce d b ya commo n mechanis m (direct o r indirect) not involvin g a^- g Availabl e dat a d o no t ten d t o suppor

t thi s hypothesis , althoug h a recen t inhalatio n toxicit ystud yo

f1,4-DC B illustrate s othe rtype s o

fdat a neede d before thes e question s ca n b e resolved . I n thi s study , significantl yhighe rlevel s o f1,4-DC B wer e foun d i n th e kidney s o f mal e rat s an d i n th e livers o ffemal e rats followin g exposur e a t 50 0 pp m fo r 2 4 hours (Umemur a e

t al. , 1990) . Althoug h th e Umemur a stud yma ysimpl y demonstrat e reactio n o f1,4-DC B wit h a -g , i

t ma yals o indicat e metaboli c difference s amon g specie s an d sexe s that influenc e th e effectiv e dose s delivere d t o th e tumo rsites . Primar ytumors i n additio n to thos e i n th e mal e rat rena l tubul e wer e no t consistentl yproduce d i n rat s o rmice a t orga n site s othe rtha n th e live r followin g administratio n o l th e eigh t chemicals . Th e productio n o ftumors a

t othe rsites , however , raise s th e possibilit ythat othe rmechanism s coul d als o b e contributin g t o th e overal l kidneytumo rincidenc e i n mal e rats . Thi s possibilit yha s bee n suggeste d fo rtetrachloroethylen e (Gree n e t al. , 1990 ; Dekan t et al. . 1989) . Dekan t an d colleague s hav e propose d a mechanis m involvin g hepati c glutathion e S conjugat e formatio n and . ultimately ,P.3 4 

# •II S 8§* § 3 — a o

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7«M e A Summary

of Genotoxkity

Data from Eight Selected

Male Rat Kidney Carcinogens Substanc e SA L AB S SC E ML A Commen U 1,4-Dichlorobenzen e Dimethy l methyl phosphonat

e

Hexachloroethan e Isophoron e

d-Limonen e Pentachloroethan e Tetrachloroethylen e

Unleade d gasolin e MLA wit h S 9 - there wa s a margina l positiv e resul t i n on e o

fthree experiments . Negativ

e i n viv o chromosom e aberrations , micronudei , an d dominan t lethals . ML A an d SC E result

s positiv e withou t S9 ; ML

A no t teste d wit h S9 ; SC E negativ e wit h S9 . AB S negativ e i n tw o labs.wit h an d without S 9 (NT P studies) , bu t positiv

e i n anothe rla b (withou t S9 ) (cite d i n NT P bioassa y report) . Drosophii a SLR L positive , but translocation s negative . Dominan t letha l positiv

e i n bot h rats an d mice . SC E reproducibl e positiv e onlywit h S9 . N o dat a fo rMLA . SC E onlypositiv

e wit h cel l cycl e dela

ywithou t S9 ; ML

A replicated positiv

e withou t S9 , no

t teste d wit h S 9 i n NT P studies . CMA a reported negatives fo r hepatocyte UDS , mouse miaonudei , an d ML A (wit h an d without S9) . Clea rnegativ

e i n al l NT P sttdies . ML A an d SC E positiv

e without S 9 (reproduced) ; SC E negativ e wit h S9 . Negativ e i n ra t i n viv o kidne yUD S assay . I n NT P studies al l clea rnegatives bot h wit h an d withou t S9 ; Also Drosophii a SLR L negative . Negativ e i n rat i n viv

o kidne yUD S assay . Positiv e i n Salmonelll a TA10 0 wit h GS H an d kidne ymicrosomes . I n NT P studies bot h positiv e responses were onlywit h S9 . Othe rstudie s confir m negativ

e response i n bacteria . I n yeast , positives hav e bee n reported fo rmitoti c recombination an d bot h positiv e an d negativ

e responses fo rgen e mutations . Negativ

e i n rat i n viv

o kidne yUD S assay . SAL - SaJmoneltaiABS-chrrxTOSomeaberratioranCHOcels; ^ assa yi n L5178 Ycells ; GS H - glutathione ; SLR L - sex-linke d recessiv e lethal : UD S - unscheduled DN A synthesis . Positiv e (+) , negativ

e (-) . ± , an d equivoca l (E ) a s define d i n Hawort h et al. . 1983 , Galowa y e t al. , 1967a , an d McGrego ret al. . 1988 . * U S . EPA Office o

fToxi c Substances submissio n b yChemica l Manufacturers Associatio n Documen t I D 40-845047 . unde rSectio n 4 o fth e Toxi c Substances Contro l Ac tP.3 5

Table 10. Summary

of Genotoxidty

Data for Two Selected Non-CIGAMale

Rat Kidney Carcinogens Substanc e SA L ABS SC E ML A Comment s Chlorothaloni l Trichloroethylen

e AB S and ML Apositiv e withou t S9 ; ML A no t teste d wit h S 9 Negativ e i n Drosophii

a SLRL. Unpublishe

d studie s negativ e i n SA L an d MLA

, a s wel l a s i n dominan t letha l and bon

e marro wcytogenetic studie

s i n mice. ML Aon variou

s catalytic fraction

s gav e mixe d results. Positiv

e UD S i n rat, mouse

, an d huma

n hepatocytes. I n viv

o studie s wit h gavag e wer e positiv e i n mouse , bu t no t i n ra t liver . Kidne y UD S i n rat s wa s negativ e (bot h gavag e and inhalation) . SA L = Salmonella ; AB

S - chromosom

e aberration

s i n CH O cells ; ML

A - thymidine-kinas

e (TKHjen e mutatio

n assa y i n L5178

Ycells ; SLRL = sex-linke d recessiv e lethal ; UD

S •unschedule

d DN A synthesis

. Positiv e (+) , negativ

e (-) , ± , and equivocal (E) a s define d i n Hawort

h e t al. , 1983 , Gallowa

y e t al. , 1987a , McGrego

re t al. , 198 8

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### , .. - w.ou o iung-iur m promotio n exposures , an d a ' sequence-reversa l studyt o discriminat e an ycocarcinogeni cfro m promo tiona l effects

. Th e test substance s were unleade d gasolin e (3 inhalatio n concentration-level s o

f10,70 , an d 30 0 ppm) , an d TM P (on e ora l dose-leve l of5 0 ppm)

. Treatmen t groups , comprise d o fapproximatel y3 0 animals , include d a control , 2 promotio n controls , a n EHE N initiatio n control , reverse sequenc e initiatio n control , initiation-promotio n grou p wit h a promotio n phas e o f2 4 weeks , initiation-promotio n grou p wit h a promotio n phas e of5 9 weeks , an d a reverse-sequence test grou p where 2 4 week s ofexposure t o unleade d gasolin e o rTM P precede d th e 2-wee k perio d o fEHE N adminis tration

. Al l animal s wer

e kille d at 6 5 t o 6 7 weeks afte rth e commencemen t ofth e experiment

. Th e result s were assesse d i n term s ofth e incidenc e of foc i o ftubul e hyperplasi a (calle d atypica l cel l foc i byth e authors ) an d rena l tubul e tumors

. Dose-relate d increase s i n hyperplasti cfoc i were observe d i n mal e rat s promote d wit h unleade d gasolin e o rTM P fo rbot h th e short - an d long-ter m promotio n periods

. Asignifican t linea rtren d i n th e incidenc e o f rena l tubul e tumor

s wit h increasin g gasolin e dos e wa s als o observe d i n mal e rat s promote d wit h unleade d gasolin e fo r2 4 week s bu t not fo r5 9 weeks

. Th e latte rdiscrepanc yreflect s a n experimenta l desig n weaknes s I n th e study , namel yunderestimatio n ofa n optima l initiatin g dos e o fEHEN , whic h resulte d i n a ver

ylo wbasa l incidenc e o

frena l tumors

. Nevertheless , th e result s wit h th e singl e dos e leve l o fTMP , an d th e absence o frena l tumors i n an ynegative contro l group , supporte d th e observe d trend s wit h unleade d gasoline

. I n th e sequence-reversa l study , there wa s n o increas e i n rena l tumors althoug h th e incidenc e o

fhyperplasti cfoc i wa s signt fteanttyelevate d fo rbot h compounds

. Foc i o

fCP N were als o score d i n thes e variou s group s wit h a n increas e upo n CIG Aexposur e apparen t i n mal e rats

. However , n o corre latio n o fincidenc e o

fCP N lesion s wit h numbers ofhyperplasti cfoc i o r incidenc e o

frena l tubul e tumor

s wa s found

. O n th e basi s o

fth e results , th e authors ' conclusion s that unleade d gasolin e an d TM P hav

e promotin g activityfo rrena l tubul e tumor s i n th e mal e rat , rathe rtha n actin g as cocarcinogens , appea rreasonable

. Furthermore , there wa s n o elevatio n o feithe rhyperplasti cfoc i o rrena l tumor s i n femal e rat s i n th e study , emphasizin g onc e again , th e mal e specificit yofth e rena l respons e t o CIGA

. Asecon d initiation-promotio n assa yusin g th e sam e EHE N mode l wa s conducte d wit h d-limonen e (Dietric h an d Swenberg

. 1991b)

. Thi s stud y specificall yaddresse d th e compariso n o fresponse s betwee n th e mal e F34 4 rat an d th e OL-g-deficlen t NB R strain

. Th e initiatin g dos e ofEHE N wa s 50 0 pp m administere d i n th e drinkin g wate rfo rtw o weeks , followe d b y d-limonen e b ydail ygavag e (5 days a week ) a t 15 0 mg/kg/dayfo r3 0 weeks

. A n initiatio n contro l (EHEN) , promotio n contro l (d-limonene) , an d a vehicl e contro l wer e include d fo rbot h strains

. I n th e F344 rat s administere d EHE N an d d-limonene , atypica l tubul e cel l hyperplasi a an d rena l tubul e adenomas were increase d 10-fol d as compare d t o th e EHE N contro l group . I n contrast , n o tumors were observe d i n anyo fth e NB R groups . Suc h negative result s 5 6 

### i n th e NB R ra t strongl ysugges t a clea rdependence o n a^- g fo rth e promotin g activit

yofd-limonene

. Th e promotiona l effec

t o funleade d gasoline , TMP , an d d-limonen e ma y b e occurrin g throug h th e influenc e o fsustaine d tubul e cel l proliferatio n whic h has bee n demonstrate d wit h thes e sam e compound s (Shor t et al. , 1989a ; Dietric h an d Swenberg , 1991b)

. Th e exten t ofcel l proliferatio n I s regarde d a s a n importan t facto ri n chemica l carcinogenesi s (Grisha m et al. , 1983 ; Cohe n an d Eltwein , 1990 ; Cunningha m et al. , 1991 ) an d stimulatio n o fcel l turnove rI s on e o

fth e ke ymechanism s believe d t o operat e I n tumo r promotio n (Farber

. 1988)

. VIII

. Compariso n o fCIG Awit h Classica l Rena l Carcinogen s I n general , classica l rena l carcinogen s o rthei ractiv e metabolite s ar e etectrophili cspecie s bindin g covalentl yt o macromolecule s an d forming , i n particular , DN Aadduct s (Hard , 1987 ; Lipsk yan d Trump , 1988 ; Alde n an d Frith , 1991)

. Suc h DN Areactivityi s putath/el yth e mechanisti cbasi s ofrena l carcinogenesi s Induce d b ythes e chemicals

. Fo rexample , carcinogeni c nitrosamine s ca n for m variou s alkylatio n product s i n DNA , includin g 0 s



### - akylguanln e whic h i s a promutageni clesio n (Pegg , 1984)

. Accordingly , classica l rena l carcinogen s are usuall ypositiv e I n short-ter m mutagenicity assays . I n contrast , CIG Aar e not know n t o reac t wit h DNAan d are generall y negative i n short-ter m test s fo rgenotoxicity

. A s describe d previousl y (Sectio n IMG) , CIG Abindin g t o ot^- g I s reversibl e an d not covalen t i n nature

. Classica l rena l carcinogen s ca n induc e rena l tubul e cance ri n rat s o r mic e i n hig h incidences , wit h minima l duratio n ofexposure , clea rdose respons e relationships , an d wit h decrease d laten t perio d o fdevelopmen t (Hard , 1987 ; Alde n an d Frith

. 1991)

. Tumo rfrequencie s ar e ofte n ove r5 0 percen t an d u p t o 10 0 percent , muc h highe rtha n th e lowincidence s (2 % t o 28 % adjusted ) recordedforCIGA

. Unlik e CIGA-induce d rena l carcinogenesis , ther e i s usuall yn o absolut e sexspecificity , wit h male s an d female s bot h susceptible , bu t sometime s t o varyin g degree

. Thes e difference s i n potency an d species - an d sex-susceptbility , suggest that classica l rena l carcino gen s an d CIG Aac

t vi a differen t mechanism s i n kidne ycarcinogenesis

. I n addition , som e classica l carcinogen s are effectiv e rena l tumo r Inducer s followin g abbreviate d dosin g regimens

. Fo rexample , DMN , DEN , an d streptozotoci n requir e onl ya singl e Injectio n t o produc e tumors , whil e th e EHE N regime n utilize s a 2-wee k perio d ofora l exposure . I n contrast , certai n militar

yfuel s Induce d renal tubul e tumors i n mal e rat s followin g lifetim e observatio n afte r1 yea ro t Intermitten t exposure , bu t no t afte r9 0 days o fcontinuou s exposur e (Bruner , 1984)

. Th e lac k o fInvolvemen t ofhyalin e drople t accumulatio n i n th e eart y nephrotoxicit

yassociate d wit h classica l carcinogen s (definit e wit h DM N an d DE N an d apparen t wit h th e others ) i s a majo rdifferenc e fro m th e sequenc e ofeart ypathologica l event s induce d b yCIG Ai n th e mal e rat

. Patholog yreport s indicat e that rena l tubul e tumor s induce d byCIG Aare morphologicall yindistinguishabl e fro m spontaneou s tumors o rthos e in 5 7

### duce d b yclassica l carcinogens , wit h bot h granula ran d clea rcel l type s

occurring

. Lfcewise , despit e difference s i n toxicityobserved , th e sequence o fdevelopmen t o fCIGA-induce d rena l tumors fro m tubul e hyperplasi a t o carcinom a appears identical

. However , som e evidenc e fro m th e bioassays suggests that th e CIG Atumors may , i n general , hav e a smalle rsize , probabl ybecaus e ofth e differenc e i n potenc ybetwee n thes e chemical s an d classica l carcinogens , affectin g th e laten t perio d oftumo rdevelopment

. As wit h classica l carcinogens , metastase s have bee n rarel yreporte d fo r rena l tubul e tumor s relate d t o treatmen t b ychemical s inducin g hyalin e droplet s and/o rot^-g

. Th e on e cas e o fmetastasi s note d wit h hexachloro ethan e suggests , however , tha t a malignan t potentia l exist s fo rsuc h neoplasms

. Althoug h th e specifi csit e o forigi n fo rth e rena l tubul e tumors produce d byCIG Ai s no t known , th e P 2 regio n o fth e proxima l tubul e a s th e primar y sit e woul d b e consisten t wit h existin g information

. Base d o n studie s wit h classica l carcinogen s thi s doe s not represen t a n unusua l location

. IX

. Evidenc e Concernin g Huma n Kidne yCance r Althoug h no t on e o

fth e most commo n neoplasm s i n th e Unite d States , rena l cel l adenocarcinoma/carcinom a i s regarde d a s a n importan t huma n cancer . Thi s i s becaus e th e diseas e i s unpredictabl e an d a significan t proportio n o

fpatients , approximatel yon e third , hav e distan t metastasi s a t th e tim e o fdiagnosi s (Benningto n an d Beckwith , 1975 ; NCI , 1987)

. Th e mortalit yrat e i n thes e case s i s high , an d overall , th e surviva l rat e fo rpatient s wit h rena l cel l cance ri s 4 8 percen t (Deves a e t al. , 1990)

. I n addition , th e etiolog yo fkidne ycance ri n human s i s poorl yunderstood

. A

. Morpholog yan d Histogenesi s Huma n rena l cel l tumors , whic h are morphologicall ysimila rt o thos e of rodents , ar e classifie d accordin g t o cel l typ e an d cellula rarrangement

. Thus , tw o mai n cel l form s ar e recognized , granula ran d clear , an d th e usua l pattern s o

forganizatio n are tubular , solid , papillary , an d cystic

. Individua l tumors ma

yshowa n admixture o

fpattern s an d o

fcel l types

. Infrequently , rena l cel l carcinom a present s a s a sarcomatoi d for m compose d o fspindl e cell s (Benningto n an d Beckwith , 1975 ; Bannaya m an d Lamm , 1980 ; Tannenbaum , 1983)

. I t i s generall yaccepte d that th e origi n ofrena l cel l carcinom a i s th e proxima l tubule , base d o n bot h immunologica l stud y(Wallac e an d Nairn , 1972 ) an d ultrastructura l feature s (Tannenbaum , 1971 ; Benningto n an d Beckwith , 1975)

. Electro n microscopyreveal s man ysimilaritie s betwee n th e tumo rcell s an d norma l proxima l tubul e epithelium , includin g brus h borde relements , membrane-associate d vesicles , an d basila rinfokJing s o f th e plasm a membran e (Tannenbaum , 1971)

. Ultrastructurally , th e amoun t o fintracellula rlipid , particulat e glycogen , an d organelle s distinguishe s clea r fro m granula rcells

. I t i s widel yconsidere d tha t huma n rena l adenomas represen t smal l adenocarcinoma s o rcarcinoma s a s there are n o microscopic , histochemi 5 8 

### cal , o rimmunologi cfeature s whic h discriminat e them , othe rtha n size , whic h i s not a n absolut e biologi cparamete r(Benningto n an d Beckwith , 1975 ; Ritchi e an d Chisholm , 1983 ; Tannenbaum , 1983)

. Adenoma s are , there fore , considere d par

t o fa n evolutionar

ycontinuu m fro m hyperplasi a throug h adenom a t o adenocarcinoma/carcinoma , a s i n rodents

. A s a genera l observation , there i s a direc t relationshi p betwee n tumo rsiz e an d frequency o fmetastasi s (Bell , 1950 ; Hellste n etal. , 1981 ; Ritchi e an d Chisholm , 1983)

. B. Incidence

and Mortality Kidne ycance rstatistic s are usuall yreporte d i n a for m whic h encom passe s al l type s o fmalignan t cance raffectin g kidney , rena l pelvis , an d sometime s urete ran d urethra

. Rena l cel l cance rrarel yoccurs unde rth e ag e of4 0 years (McLaughli n an d Schuman

. 1983 ; Asa l et al.

. 1988a ) an d represent s abou t 7 0 percen t ofal l kidne ytumor s i n adult s (Deves a e t al. , 1990)

. Kidne ycance rstatistics , therefore , provid e a n approximatio n onl yo f rena l cel l tumo rprevalence

. Th e numbe rofnewcase s o fkidne yan d urinar

ytract cance r(excludin g bladder) estimate d fo r199 1 i n th e U S i s 25,30 0 wit h a mortalityestimat e of 10,60 0 death s (Borin g e t al. , 1991)

. Thes e figure s represen t approximatel y 2 percen t o fbot h newcance rcase s a t al l site s an d tota l cance rdeaths

. Th e age-adjuste d incidenc e rate s i n th e U S fo rth e perio d betwee n 197 5 an d 198 5 obtaine d fro m th e NC I Surveillance , Epidemiolog yan d En d Result s Progra m (SEER ) dat a fo rrena l cel l cance rar e 8. 4 pe r100,00 0 fo rmale s an d 3. 7 pe r100,00 0 fo rfemales , wit h n o differenc e amon g racia l group s (Deves a etal. , 1990)

. Most studie s indicat e a consisten t mal e t o femal e rati o of2: 1 forth e incidence o

frena l cel l tumor s (Asa l etal.

. 1988a ; Deves a etal.

. 1990)

. I n considerin g rena l cel l tumor s specifically , th e highes t rate s interna tionall yhav e bee n reporte d fro m Icelan d an d othe rScandinavia n countries

. Rena l cel l carcinom a i s th e fift h mos t commo n malignan t tumo ro fmale s i n Icelan d althoug h i t rank

s onl ytent h i n female s (Thorhallso n an d Tulinius , 1981)

. Th e lowest rate s fo rrena l cance rar e recorde d i n Africa , Asia , an d Sout h Americ a (McLaughli n an d Schuman , 1983)

. Withi n th e US , mortalit y survey s indicat e that th e Nort h Centra l regio n an d som e area s i n th e Northeas t hav e th e highest incidenc e rat e fo rrena l cel l carcinom a (Pickl e e t al.

. 1987)

. I t ha s bee n suggeste d tha t th e clusterin g i n th e Nort h Centra l regio n mayb e partiall yexplaine d b yth e predominantl yGerma n an d Scandinavia n origi n o

fth e area s populatio n (McLaughli n et al. , 1984)

. Severa l studie s reporte d that th e urba n rate s fo rrena l cel l tumo rincidenc e are highe rtha n fo rrura l areas , bu t th e correlatio n i s considere d t o b e wea k (Newso m an d Vugrin , 1987)

. I n contras t t o th e relativel ylo wincidenc e an d mortalityfigure s fo r malignan t kidne yan d relate d tumors provide d b ycance rstatistic s data , th e occurrenc e o

frena l cel l adenomas a

t autops yi s common . Th e reporte d incidenc e has range d fro m 1 5 percen t (Bannaya m an d Lamm , 1980 ) t o 2 5 percent , th e latte rfo rmale s ove rth e ag e of5 0 (Rees e an d Winstanley , 1958)

. Thes e finding s have le d t o speculatio n tha t a proportio n ofadenoma smayreac h a limit o fgrowt h and/o rremai n quiescen t (Bannaya m an d Lamm , 1980 ; Warier , 1983) . Durin g th e perio d 195 0 t o 1985 , th e U S Cance rStatistic s dat a indicate d a n increase o

f8 2 percen t i n th e incidenc e o fkidne yan d rena l pelvi s cance r combine d (NCI , 1987) . Fo rrena l cel l cance ralone , th e increas e amon g whites , base d o n compariso n o fdat a fro m 196 9 t o 197 1 wit h dat a fro m 198 3 t o 1985 . wa s abou t 3 0 percent ; thi s woul d b e a n averag e annua l percen t chang e i n incidence o

f2. 0 fo rmale s an d 1. 8 fo rfemale s (Deves a e t al. , 1990) . Dat a fro m Cance rRegistrie s i n Scotlan d als o indicate d a n increase d incidenc e o

frena l cel l carcinom a o fapproximatel y3 7 percen t fo rmale s betwee n 196 7 an d 1979 , althoug h ther e wa s n o correspondin g increas e i n female s (Ritchi e an d Chisholm , 1983) . Despit e a n improvemen t i n mortality rate s since 195 0 compare d t o incidenc e rate s (NCI , 1987) , th e relativ e 5 - yea rsurviva l rates , whic h are clos e t o 5 0 percent , have not altere d sinc e th e earty 1970 s (Borin g et al. , 1991) , suggestin g littl e improvemen t i n treatmen t ove rth e past two decades . O n th e othe rhand , diagnosti c detectio n measure s have improve d dramaticall ydurin g thi s tim e whic h ma yexplain , a t least i n part , th e observe d increas e i n rena l cance rincidenc e (Higginso n e t al. , 1984 ; NCI , 1987) . Rena l cel l carcinom a ha s bee n diagnose d wit h increasin g frequenc y i n patient s wit h chroni c rena l failur e (Hughso n et al. , 1986 ; Newso m an d Vugrin , 1987) . I n particular , thi s appear s t o reflect a n associatio n wit h th e developmen t o

facquire d rena l cysti c diseas e whic h frequentl yoccur s i n patient s o n long-ter m hemodialysis . Th e incidence o frena l cel l carcinom a i n patient s wit h acquire d cysti c diseas e ha s bee n estimate d a s approxi matel y6 percen t (Hughso n e t al. , 1986) . Thus , curren t dat a sugges t tha t a growin g populatio n o

fhuman s receivin g maintenanc e dialysi s ma yb e a t ris k fo rdevelopin g rena l cel l tumors . C. Environmental

and Lifestyle

Factors Potentia l etiologica l association s betwee n rena l cel l cance ran d exog enou s an d endogenou s environmenta l factors , lifestyle , an d occupation , hav e bee n sough t i n cohor t an d case-contro l studies . O fal l th e environmen ta l an d lifestyl e factors investigated , tobacc o us e i n th e for m o fcigarette , cigar , o rpip e smokin g ha s bee n th e on e mos t consistentl yassociate d wit h rena l cel l carcinom a (Daya l an d Kinman , 1983 ; McLaughli n an d Schuman , 1983 ; Y u etal. , 1986 ; Asa l etal. , 1988a ; Brownson , 1988 ; L a Vecchi a etal. , 1990) . Althoug h a fewstudie s hav e faile d t o identif ya statistica l associatio n betwee n smokin g an d rena l cel l cancer , i t ha s bee n estimate d tha t 3 0 percen t o

frena l cel l carcinoma s i n male s an d 2 4 percen t i n female s ma yb e attributabl e to cigarett e smokin g (McLaughli n e t al. , 1984 ) an d that ther e i s evidenc e fo ra moderate dos e respons e (McLaughli n an d Schuman , 1983) . On e stud yhas als o linke d us e o fchewin g tobacc o wit h rena l cel l carcinom a i n male s (Goodma n e

t al. , 1986) , an d anothe rha s associate d smokin g wit h rena l adenom a (Benningto n e t al. . 1968) . Othe rpossibl e ris k

factor s that hav e bee n reporte d includ e coffe e an d te a consumption , artificia l sweeteners , hig h bodymas s index(maintaine d 6 0 fro m 2 0 year s o fage) , hig h dietaryanima l protei n an d fat , lowe reducation ; levels , long-ter m analgesi c use . an d diuretic s (reviewe d i n Daya l a n Kinman , 1983 ; McLaughli n an d Schuman , 1983 ; McLaughlin , 198 ' McLaughli n e

t al. . 1984 ; Goodma n et al. . 1986 ; Y u e t al. , 1986 ; Asa l e t a l 1988a ; McCredi e e

t al. , 1988) . O fthese , th e evidenc e i s leas t consisten t k beverag e consumption , artificia l sweeteners , othe rdietaryfactors , a n socioeconomi c status , an d i s strongest fo rhig h bod ymas s inde xan d dr u us e (phenaceti n an d diuretics) . D. Occupational

Factors Althoug h a numbe ro

fepidemiologica l studie s hav e reporte d so m associatio n betwee n occupatio n an d rena l cancer , clea roccupation , determinant s hav e ye t t o b e demonstrate d an d i t i s considere d tha t mu c epidemiologica l researc h i s neede d t o furthe rdefin e an d quantif ypotentb risks (McLaughli n an d Schuman , 1983) . Occupationa l exposure s i n Nor t America , wher e at leas t on e stud yha s reporte d a n associatio n wit increase d kidney cance rrates , includ e asbesto s (Selikof fet al. , 1979 ; Smit e t al. , 1989) , coke-ove n emission s i n th e stee l industr y(Redmon d e

t a l 1972) . printin g pres s chemical s (Paganini-Hil l et al. , 1980) . laundry- an d dn , cleanin g agent s (Blai re t al. , 1979 ; Kat z an d Jowett , 1981 ; Du h an d As a 1984 ; Asa l et al. , 1988b) , exhaus t fume s i n truc k driver s (Brownson , 198 8 petroleum , tar , an d pitc h products (Thoma s e t al. , 1980 ; Hani s e t al. , 198 2 We n e t al. , 1983 ; McLaughli n et al. , 1984 ; Savit z an d Moure , 198 ' Kadaman i et al. , 1989) , an d aviatio n an d jet fuel s (Siemiatyck i et al. , 198 7 I n thes e studies , informatio n o n smokin g histor ywa s rarel yavailable , so th« its possibl e influenc e coul d no t b e determined . A stud ythat examine d th e relationshi p betwee n rena l cance ra n occupatio n a s define d i n th e 196 0 Censu s i n Sweden , wher e th e inciden c rate s ar e highe rtha n i n th e US , di d no t detec t increase d ris k fo rheart h a n furnac e worker s i n th e stee l industry , printin g workers , laundr yan d dr y cleanin g workers , o rworker s i n petroleu m refinerie s an d gasolin e statio n (McLaughli n e t al. , 1987) . Instead , th e Swedis h stud yreporte d a n increas i n incidenc e o

frena l cel l cance ramon g healt h car e professionals . E. Renal

Cancer and Hydrocarbon, Solvent or Petroleum

Product Exposure Severa l o

fth e occupation s liste d abov e involve exposure to certai i classe s o

fchemical s that mayfal l into th e CIG A category . Beside s CIG A however , non-CIG A compound s are als o present , makin g i t difficult t < attribut e elevation s i n risk wit h a uniqu e exposur e (e.g. , CIGA) . I n a rece n population-base d case-contro l study , Kadaman i et al . (1989 ) di d no t o b serv e statisticall ysignifican t association s betwee n rena l cel l carcinom a an t hig h occupationa l exposure t o hydrocarbon s i n male s (O R 1.6 ; 95 % C I 0. 7 3.6 ) o ri n female s (O R 0.8 ; 95 % C I 0.3-2.3) . Th e authors , however , note * a positive exposure-respons e relationshi p fo rthos e wit h olde rage s an d to worker s wit h th e greates t duratio n o fexposure . I n a case-referen t stud yo foccupationa l ris k

indicators fo rrena l cel l adenocarcinomas , Partane n et al . (1991 ) examine d al l case s reported ir j 6 1Finlan d i n 197 7 t o 1978 . Thes e investigators foun d a n elevate d ris k an d a n exposure-respons e relationshi p fo rgasolin e exposure . Sinc e th e postu late d averag e latenc yperio d wa s abou t 3 0 years , a rol e fo rlea d compound s coul d no t b e rule d out . Syntheti c solvent s widel yuse d i n dry-cleanin g includ e th e CIGA , tetrachloroethylene , a s wel l a s Stoddar d an d 140 F solvents. 4 Severa l studies analyzin g proportiona l mortalit ydat a o n laundry - an d dry-cleanin g workers i n variou s part s o fth e U S reporte d elevate d risk s fo rkidneycance r (Blai re t al. . 1979 ; Kat z an d Jowett , 1981 ; Du h an d Asal . 1984 ; Asa l e t al. , 1988b) . Mor e recen t studie s that wer e bette rdesigned , however , have no t substantiate d th e earlie rfindings . N o statisticall ysignifican t elevation s i n kidne ycance rrisk

s hav e bee n detecte d i n th e studie s o fdry-cleanin g workers b

yBlai re t al . (1990 ) (Standardize d MortalityRati o [SMR j 50 ; 95 % C I 10-180) , Lyng e an d Thygese n (1990 ) (Standardize d Incidenc e Rati o |SIR)males, 1 5;95%CI 0 6- 3 3;lemales 0 6.95%CI0.2-1.4),orBrownan d Kapla n (1987 ) (SM R 200 ; 95 % C I 55-517) . I n considerin g occupationa l exposure t o solvent s a s a genera l chemica l category , Harringto n e t al . (1989 ) foun d n o relationshi p wit h rena l cance r(O R 1.0 ; 95 % C I 0.2-4.9 ) althoug h th e statistica l powe ro t thi s study , a s wit h most others , wa s acknowledge d b yth e authors a s sufficien t t o identif yonl ylarg e ris k

esti mates . Siemiatyck i et a l (1987 ) conducte d a population-base d case-referen t studyi n Montrea l o n cance rassociation s wit h exposur e t o 1 2 petroleum derive d liquids . Thes e variou s mixture s include d automotive an d aviatio n gasoline s an d distillat e jet fuel . Aviatio n gasolin e differ s i n compositio n fro m th e automotive counterpar t b yits hig h conten t o

falkylat e naphthas , consti tute d mainl yo

fbranche d alkane s (Siemiatyck i e t al. , 1987) . N o statisticall y significan t ris k

o frena l cance rwa s foun d wit h exposur e t o automotiv e gasolin e (O R 12 ; 90 % C I 0.8-1.6) . Statisticall ysignifican t elevations , however , wer e note d a t th e 9 0 percen t confidenc e leve l wit h exposure t o aviatio n gasolin e (O R 2.6 ; 90 % C11.2-5.8 ) an d t o je t fue l (O R 2.5 ; 90 % C I 1.1-5.4) . Si xo

fth e seve n case s wit h exposure t o aviatio n gasolin e als o ha d exposure t o jet fuel , makin g i t difficul t t o distinguis h a uniqu e exposure . I n dept h analyse s o

fth e tw o association s usin g logisti c regressio n method s indicated , however , a greate rrol e fo raviatio n gasolin e tha n fo rje t fuel . Won g an d Raab e (1989 ) conducte d a quantitative meta-analysi s by cance rsite o

fpetroleu m industr

yemployee s fro m th e US , Canada , Unite d Kingdom , Europe , Australia , an d Japan , criticall yreviewin g almost 10 0 publishe d an d unpublishe d epidemiologica l reports . Standardize d mortalit y ratio s observe d fo rkidneycance ri n th e industr ya s a whol e wer e simila rto those fo rth e genera l population . Result s fro m refiner y studie s range d fro m nonsignifican t deficit s t o nonsignifican t excesses . However , th e possibilit y o fa n elevate d kidneycance rris k wa s raise d fo ron e specifi c grou p withi n th e

Stoddar d an d 140 F solvent s ar e mixtures o l hydrocarbon s includin g straigh t an d branche d chai n paratlin s suggestin g tha t the yma yals o b e CIGA . industry . Drivers amon g Britis h distributio n worker s (Rushto n an d AkJerson , a s reporte d b

yWon g an d Raabe ) showe d borderlin e significanc e fo rexces s kidneycance rmortality . Won g an d Raab e (1989 ) conclude d that additiona l data , particularl yinvolvin g expos u r e t o downstrea m gasoline , ar e neede d t o resolve th e issue . I n a larg e population-base d case-contro l stud yadjuste d fo rth e con foundin g factors o

fag e an d cigarett e smoking , n o overal l associatio n (O R 1.0 ; 95 % C I 0.7-1.4 ) wa s observe d betwee n ris k fo rrena l cel l cance ran d employmen t i n a rang e o foccupation s wit h potentia l fo rexposur e t o petroleu m products (McLaughli n e t al. , 1985) . Ther e was , however , a smal l exces s ris k

amon g gasolin e statio n attendant s (O R 1.2 ; 95 % C I 0.6-2.3 ) whic h increase d wit h duratio n o femployment , althoug h individua l poin t estimate s an d test s fo rtrend s wer e not statisticall ysignificant . A case contro l stud yo n a combine d cohort o fapproximatel y100,00 0 mal e refinery worker s fro m five petroleumcompanies , sponsore d b y th e America n Petro leu m Institute (Pool e et al. , 1990) , suggeste d increase s i n kidneycance rrisk fo rlaborers (Relativ e Ris k (RR J 1.9 ; 95 % C11.0-3.9) , worker s i n receipt , storage , an d movement s (R R 2.5 ; 95 % C I 0.9-6.6) , an d refineryuni t cleaner s (R R 2.3 ; 95 % C I 0.5-9.9 ) whe n compare d wit h a referenc e grou p o foffic e workers , professionals , an d technicians . I n th e cohor t ther e wer e 10 2 kidneycance rcase s amon g 18,32 3 deaths . I n evaluatin g unleade d gasoline . 5 5 relevan t studie s wer e reviewe d by th e USEP A (1987 ) to determin e whethe rther e wa s an yepidemiologi c evidenc e fo ra n associatio n betwee n gasolin e exposure an d cance rrisk . Th e evidenc e fo rdrawin g causa l inference s betwee n unleade d gasolin e an d cance rwa s considere d inadequat e unde rEPA' s guideline s fo rcance r risk assessmen t (USEPA , 1986) . A s Enterlin e an d Vire n (1985 ) hav e emphasize d i n thei rrevie wo n th e epidemiolog yo frena l cance ran d gasolin e exposure , mos

t o fth e studie s hav e not bee n designe d o ranalyze d wit h a n hypothesi s specificall yassociatin g gasolin e exposure an d rena l cancer . Th e cohor

t studie s o

fpetroleu m worker s d o no t len d themselve s fo ra compariso n sinc e theyshe d n o ligh t o n gasolin e exposure , pe rse . Expo sure s i n thes e studie s hav e bee n varied , an d th e onl ycommo n elemen t i s th e plac e o

fwork . Thus , th e individual s i n th e cohor t wh o ha d th e exposur e o finterest , i.e. , gasolin e o ra specifi c fraction , cannot b e identified . A s a genera l conclusio n fro m th e foregoing , smal l risk s canno t b e exclude d fo rspecifi c jo b categories , bu t th e associatio n betwee n huma n kidneycance ran d exposure t o petroleu m distillates , i fther e i s one , doe s not suggest hig h risk

s to rth e type s o

fexposure s that hav e occurre d i n th e past . X . Evidenc e fo rDose - an d Time-Dependen t Progressio n fro m Earl yt o Lat e Lesion s A n importan t aspect to rexaminin g th e hypothesi s that rena l tumo r formatio n i s directl yassociate d wit h accumulatio n o fot^- g i n th e mal e rat kidneyi s a demonstratio n o fth e progressio n o

flesion s propose d to culminate i n neoplasia . Fo rsom e o fth e steps , clea rdose-respons e relationship s have bee n shown . I n othe rcases , histopathologicall yobserv -abl e events are o fa secondarynature an d no t i n th e direct progression . A s th e followin g informatio n shows , however , dat a demonstratin g th e existenc e o fothe rsteps i n th e propose d progressio n are limited , restrictin g confidenc e i n judgments o n th e natur e o fth e association . Evaluatio n o

fth e event s leadin g to neoplasi a i s furthe rcomplicate d by th e lo wincidenc e o

frena l tumors induce d b yth e CIGA studied . Suc h Informatio n make s i t difficul t t o identif ypossibl e relationship s betwee n th e induce d nephropath yan d rena l carcinogenesis . A . Associatio n Betwee n CIGA , Hyalin e Drople t Formation , an d Alpha^-globull n Accumulatio n Dose-dependen t relationship s have bee n demonstrate d betwee n th e administratio n o

fd-limonen e (Lehman-McKeema n e t al. , 1989 ) o rgabapenti n (Dominic k

et al. , 1990 ) an d excessive formatio n ofhyalin e droplets , an d betwee n unleade d gasolin e o rTM P an d o_- g accumulatio n (Olso n e t ai. , 1987 ; Charbonnea u et al. , 1987) . I n th e d-limonen e study , hyalin e droplet s wer e grade d o n a scal e o f0 t o 1 2 accordin g to size , eosinophili c intensity , an d th e numbe ro

ftubule s loade d wit h droplets . Th e droplet score s fo r d-limonen e dose s o

f0,0.1,0.3,1.0 . an d 3. 0 mmol/kg were , contro l t o hig h dose . 3.4.5 . ca.7,8 , an d 1 0 (Lehman-McKeema n e t al. , 1989) . Th e dose respons e relationshi p wit h OL- g accumulatio n i s exemplifie d bymeasure ments followin g administratio n o fTMP , which , give n at singl e dose s o f 0.044,0.440 , an d 4.00 0 mmol/kg . induce d ot^- g concentration s i n rat kidney tissu e at 2 4 hour

s o f10.3.17.3 , an d 28. 1 mg/ g wet weight , respectively , against a contro l valu e o f9. 5 mg/g we t weigh t (Charbonnea u e t al. , 1987) . Wit h orall yadministere d gasoline , th e OL- g concentration s wer e dose responsiv

e onl yi n th e rang e o

f0.0 4 to 1.0 0 mL/kg (Olso n et al. , 1987) . I n a specia l NT P study , mal e an d femal e F34 4 rat s wer e expose d t o d-limonen e bygavag e fo r1 4 day s ove ra 21-da y perio d (NTP , 1990) . Th e ot^- g content , quantitate d wit h a n ELIS A test i n kidneyhomogenates , increase d significantl y i n dose d mal e rats relative t o vehicl e controls . A t 7 5 mg/kg , th e lo

wdos e employe d fo rmal e rat s i n th e 2-yea rbioassay , OL- g level s wer e approximatel ydoubl e thos e i n controls . I n females , increasin g th e dos e a s hig h a s 1,20 0 mg/k g ha d n o measurabl e effect o n ot^- g level s i n th e kidney . Althoug h microscopi c examinatio n o fkidneysection s staine d wit h hematoxyli n an d eosi n ( H & E ) showe d n o visibl e difference s betwee n dos e an d vehicl e contro l mal e rats , i n plasti c embedde d section s staine d wit h Lee' s methylen e blu e basi c fuchsin , difference s i n th e distribution , amount , an d shap e o

fintracytoplasmi c granule s i n th e proxima l tubule s wer e detected . I n contrast t o th e 21-da y follow-u p study , th e 13-wee k range-findin g stud yconducte d before th e d-limonen e bioassayfaile d t o detec t a n accu mulatio n o

fhyalin e droplets . Th e NT P repor t (1990 ) acknowledge d that thi s failure migh t have bee n relate d t o th e fact that severa l day s passe d betwee n th e tim e th e chemica l wa s last administere d an d th e tim e th e animal s wer e kille d fo rhistologica l examination . Othe rstudies hav e show n tha t rena l ct^- g concentration s declin e rapidly , reachin g preexposur e level s byth e 6 4 thir d da y afte rtreatment , althoug h hyalin e droplets , bein g structura l entities , require u p t o 9 days fo rcomplet e resolutio n (Gar g et al. . 1988) . Thi s suggest s that th e interva l betwee n th e tim e th e chemica l wa s las t adminis tere d an d th e tim e th e animal s wer e kille d fo rhistologica l examinatio n i s critica l to findin g hyalin e droplet s an d probabl yaccount s fo rdiscrepancie s foun d amon g som e studies . Thes e variou s observations , alon g wit h th e result s o t ct^- g localizatio n studie s an d bindin g studie s considere d earlier , suppor t a causa l associatio n betwee n th e administratio n o fCIG A an d ct^- g accumulatio n i n hyalin e droplets . B. Association

Between

Hyaline Droplet Formation, Cell

Necrosis

and Tubule Cell Regeneration Hyalin e droplet accumulation , single-cel l necrosis , an d cel l proliferatio n occu rpredominantl yi n th e P 2 segmen t ofth e proxima l tubul e followin g CIG A administratio n (Shor t et al. , 1987 , I989a,b) . Althoug h single-cel l necrosi s ha s bee n clearl ydemonstrate d i n associatio n wit h cellula rhyalin e droplet accumulatio n (Kanerv a et al. , 1987a ; Short e t al. , 1987) , ther e are n o dose-respons e studie s quantitatin g th e relationshi p betwee n increase d hyalin e droplet s an d cel l necrosi s i n histologica l sections , o rbetwee n cel l necrosi s an d cel l regeneration . However , Alde n ha s show n a correlatio n betwee n th e hyalin e droplet response , increase d mitoti c indexi n proxima l convolute d tubules , an d elevatio n o fth e numbe ro

fcell s excrete d hourl yi n th e urin e (a n indexo

fexfoliate d necroti c tubul e cells) , usin g two dos e level s o fd-limonen e give n orall yfo r3 week s (Alde n an d Frith , 1991) . Dose-respons e relationship s betwee n hyalin e droplet accumulatio n an d proxima l tubul e cel l proliferatio n hav e bee n observed . Shor t an d coworker s (1987 ) expose d mal e rat s fo r3 week s t o TM P (oral ) o runleade d gasolin e (inhalation ) an d the n measure d [ 3 H J thymidin e labelin g indices . Th e exten t an d severityo fhyalin e droplet accumulatio n parallele d th e exten t an d localizatio n o

fcel l proliferatio n i n proxima l tubul e cells , an d bot h parameter s wer e increase d indose-dependen t fashio n (Figure s 4 an d 5) . I n a n extende d stud yo

fth e sam e compounds , Short e t al . (1989a ) observe d 6 - t o 1 1 -fol d increase s i n labelin g indice s i n th e P 2 segmen t o fth e ra t kidne y afte rth e rat s receive d 3,10 , an d 2 2 week s o fexposur e t o 30 0 pp m unleade d gasolin e o r5 0 pp m TMP . Thes e labelin g indice s remaine d 4 - t o 6-fol d highe r tha n contro l value s durin g th e 48t h wee k ofexposure . I n contrast , Via u et al . (1986 ) di d not observ e a sustaine d regenerativ e respons e i n th e kidney s o fmal e rat s expose d t o a n isoparatlini c solvent consistin g o

fsaturate d C, 0 -C „ aliphati c hydrocarbon s beyon d 5. 5 weeks . Labelin g indice s i n th e cortexo t treate d rat s at 4 6 an d 6 8 weeks were n o differen t fro m th e controls . Thi s apparen t discrepanc ybetwee n th e gasolin e an d TM P result s undoubtedl yreflect s difference s i n th e techniqu e o f radioactive labeling . Via u e t al . (1986 ) use d a singl e injectio n o fFH) - thymidin e 1 hou rprio rto sacrifice , whereas , Shor t et al . (1989a ) labele d continuousl yb

ysubcutaneou s osmoti c minipum p infusio n ove ra 7-da y period , th e preferre d metho d fo rcel l population s wit h a lo wcel l turnover , thereb yincreasin g th e amoun t o fradioactivityincorporate d int o rena l tissue . 6 5V

o »> t > o c V u a > 0 .

1 1 0 Dose TM P (mg/kg ) 10 0 0. 1 1 1 0 10 0 100 0 

## Dos e Gasolin e (ppm )

FIgureS: Dose-response relationship

between

renal hyaline droplet accumulation

(D) and f>H] thymidine

labeling

index of proximal

tubule P2 cells in male F344 rats gavaged with TMP tor 5 consecutive

days per week lor 3 weeks. Seven-day osmotic minipump

implanted

on twelfth day after start of dosing. Rats

killed and fixed on 22nd day. Source: Adapted

from Short et al.. 1987. Figure 5: Effect

of 0 to 2.000 ppm unleaded

gasoline

on continuous

uptake of /*H/ - TdR by PI, P2. and

P3 segments

of the proximal

tubule epithelium. Each

test point is the mean value determined

from 3 rats. Dosage

is presented

on a log scale. Source: Adapted

from Short et al.. 1987.

6 6 6 7

### Irrrecover ystudie s wit h unleade d gasolin e an d TMP , Shor t an d coworker s (1989a) showe d tha t neithe rincrease d hyalin e droplet s norcel l prolifera tio n were observabl e 7 day s afte rdiscontinuin g th e 3-wee k exposures , indicatin g complet e recovery . However , afte r1 0 an d 2 2 wee k period s of exposure , recover

ywa s onl ypartial , labelin g indice s remainin g nearl ythre e time s above control s followin g 1 0 days i n a gasoline - o rTMP-fre e environ ment

. Thus , proxima l tubul e cel l proliferatio n i s a persisten t phenomeno n i n chroni cexposure t o CIGA , becomin g les s amenabl e t o recover ywit h increasin g duratio n o

fexposure . Furthermore , i n promotio n studie s wit h d-limonene , cel l proliferation , assesse d b ybromodeoxyuridin e labelin g vi a subcutaneou s osmoti c minipum p implants , wa s not induce d beyon d backgroun d byd-limonen e afte r5 o r3 0 weeks o

fexposure i n th e O L-g-deficien t NB R rat , compare d t o a 5-fol d increas e i n th e tubul e cel l labelin g of d-limonene-promote d F34 4 rats initiate d wit h EHE N (Dietric h an d Swenberg , 1991b)

. Thi s resul t suggests that th e sustaine d proliferativ e respons e induce d b ya CIG Ai s dependen t o n th e ot^- g syndrome

. Thus , th e sequenc e o

f event s followin g CIG Aadministratio n involve s lysosoma l overload , cel l necrosis , an d cel l replication

. Al l thre e of thes e occu ri n th e sam e segmen t o fth e nephro n i n conventiona l strain s ofrats , bu t non e occu ri n th e NB R rat

. Whereas thes e events are temporall ycorrelated , i t i s no t yet clea rwhethe rth e lysosoma l overloa d cause s necrosi s o rwhethe r necrosi s ca n b e linke d wit h replication

. Thes e question s nee d furthe r investigatio n an d hypothesi s developmen t i n orde rt o establis h mechanism s o faction

. C. Progression

to Cast Formation, Tubule

Dilation, and

Mineralization Sinc e fe wchroni cstudie s incorporate d seria l sacrifices , i t i s difficul t t o asses s th e time-dependenc e o f th e developmen t an d progressio n of th e sequentia l lesion s propose d t o b e associate d wit h ot^- g nephropathy

. Granula rcas t formatio n wa s recorde d exclusivel yi n mal e rat s fo rmost o fth e selecte d chemical s evaluate d i n 13-wee k toxicitystudie s b yth e NT P an d sometime s i n th e 2-yea rbioassays

. I n anothe rstudy

. Via u e t al

. (1986 ) expose d mal e rat s t o C, 0 

### -C 1 2 

### aliphati chydrocarbon s b yinhalatio n fo r5.5,46 , o r6 8 weeks an d foun d granularcast s a t th e earlies t time-point , bu t the ywere absen t a t th e late rtime-points

. On e explanatio n fo rthes e result s i s that certai n lesion s i n th e sequenc e ar e transitor

yi n nature

. Granula rcasts , fo r example , ar e assume d t o b e linke d t o th e activ e hyalin e drople t overload

. Once a^- g level s becom e lowbecaus e o f age , afte rapproximatel y1 6 months , th e numbe ro

fnewhyalin e droplet s bein g forme d shoul d becom e minimal

. Asecon d explanatio n i s that subtl e change s suc h a s granula rcas t formatio n an d th e associate d tubul e dilatio n ca n b e obscure d b yth e developmen t of CP N i n late rstages

. Tubul e dilatio n i s presume d t o follo wobstructio n ofth e nephro n b yth e accumulatio n o

fgranula rcast s compose d o fsloughe d epithelia l cel l debri s i n th e tubul e lumen

. Figure 6 shows on e exampl e o fth e interrelationship s 6 8 

### =5 = Hyalin e Droplet s =TO =

Regeneratin g Epitheliu m

0 MrA 0 Tubul e Dilolio n ——A 1 2 3 

### Week s o f treatmen t

«„,, , « •Time-seouence for the development

of hyaline droplets, regenerating tui " ^ eZL^a^tuboleMat^ unleaded

gasoline

daily by gavage for a 28 day penod. Source: Adapted

from Thomas

etal., 1985.

6 9

### observe d betwee n hyalin e drople t formation , epithelia l cel l proliferation , an d

tubul e dilation

. I n thi s study , mal e rat s were administere d unleade d gasolin e ( 2 g/kg/day

) fo ra perio d o

f2 8 days an d examine d a t 5 interi m time-point s (Thomas etal. , 1985)

. A n initia l accumulatio n o fhyalin e droplets , commenc in g o n th e firs t da

yo fexposure an d persistin g throughout , wa s followe d at 14,21 , an d 2 8 days , byincrease s i n epithelia l cel l proliferatio n an d tubul e dilatio n associate d wit h lumena l accumulatio n o fgranula rdebris

. Linea rmineralizatio n i n th e rena l papill a o fmal e rats ha

s bee n consis tentl yobserve d i n a numbe rofNT P an d othe r2-yea rbioassays wit h potentia l CIG Acarcinogen s bu t not i n th e 13-wee k toxicit ystudies

. Clea r dose-respons e relationship s were demonstrate d fo r1,4-DC B (NTP , 1987a) , JP- 4 mixe d distillat e (MacNaughto n an d Uddin , 1984) , an d unleade d gasolin e (USEPA , 1987)

. I n th e 2-yea runleade d gasolin e stud yther e were interi m sacrifice s a

t 3 , 6.12 , an d 1 8 month s permittin g quantitativ e obser vatio n o n th e incidence ofmineralizatio n (USEPA , 1987)

. Althoug h thi s lesio n was terme d pelvi crathe rtha n medullar ymineralizatio n i n th e origina l repor t fro m IRDC , i t was qualifie d a s referrin g t o materia l locate d withi n lubule s o fth e rena l pelvis , thu s conformin g t o th e medullar ysit e see n wit h otherCIGA

. Tabl e 1 1 present s a summar yofthes e dat a whic h shows a clea r dose-relate d progressio n i n th e incidenc e o fmineralizatio n fro m 6 month s u p to

. an d including , th e 2-yea rsacrifice

. Paralle l dose-respons e increase s hav e bee n demonstrate d fo rmedullar ymineralizatio n an d urothelia l hyperplasi a wit h JP- 5 je t fuels , Diese l Fue l Marine , an d decali n (Brune r 1986) , supportin g th e notio n that th e pelvi chyperplasi a i s a urothelia l respons e t o mineralizatio n i n th e papilla

. D

. Associatio n Betwee n CIG Aan d Chroni cProgressiv e Nephropath y Althoug h exacerbatio n o fspontaneou s CP N b yCIG Ahas bee n note d i n man ystudies , quantitatio n o fthi s respons e has bee n attempte d o n fe w TmHI- n Table 11. Incidence

of Medullary

Mineralization

in Male Rats During Inhalation Exposure

to Unleaded

Gasoline Observatio n time-point s (months ) 3 6 1 2 1 8 2 4 Exposur e level s o funleade d gasolin e vapo r(ppm )

'Th e incKlenc e olmedu ^ mineralJMtK) n j s ^ ^ Source Adapte d from USEPA , t 9 8 7 7 0 0 6 7 

## ~° ~~o 0 0 0 0 0 0 

## ° 5 29 2 0 0 2 0 2 0 6 3 205 6

0 20 * 8 0 8 0 9 1 a s percen t o l animal s affec^ec T 

### occasions

. Shor t etal

. (1989a ) compare d th e numbe ro fCP N foclperkidne y sectio n i n mal e rat s at thre e dos e level s o funleade d gasolin e exposur e an d tw o chroni ctime-points , wit h contro l specimens

. Fo ra dail ydos e rang e o f 0,10,70 , an d 30 0 pp m unleade d gasoline , th e numbers offoc i observe d a t 2 2 week s ofexposure wer e 0.4 , 0

. 1.0 , an d 6.3 , respectively , an d at 4 8 week s o fexposure , 5.0,4.0,10. 0 an d 9.0

. Thi s study , therefore , support s th e conclusio n that ther e i s a n earlie ronse t ofCPN

. demonstrabl e b y5 months , an d a highe rincidenc e o fdiseas e i n th e middle - an d high-dos e groups . I n th e NT P bioassayo fd-limonen e (NTP , 1990) , treate d mal e rat s showe d a spectru m ofcompound-relate d kidne ylesions , includin g exacerbatio n ofCPN

. mineralizatio n i n th e rena l medulla , hyperplasi a o t th e epitheliu m linin g th e rena l papilla , an d proliferativ e lesion s o fth e rena l tubul e epithelium

. Th e severityofCP N o n a scal e of0 t o 4 wa s grade d a s "not present , minimal , mild , moderate , o rmarked. " Th e mea n valu e increase d wit h increasin g d-limonen e dos e fro m 1. 5 i n vehicl e control s t o 1. 8 an d 2. 2 i n animal s dose d a t 7 5 an d 15 0 mg/kg , respectively

. A s CP N i s exacerbate d b yCIG Aadministration , an d CPN-affecte d tubule s have a hig h cel l turnove rrate , i t ha s bee n suggeste d tha t CPN may playa rol e i n rena l tumo rproductio n followin g a.- g nephropath ybecaus e enhance d regeneratio n i s considere d a risk facto rfo rcarcinogenesi s (Trum p et al.

. 1984b ; Shor t e t al. , 1989b)

. There i s n o fir m evidenc e availabl e t o dat e that substantiate s o rdisprove s a lin k betwee n C P N an d rena l tubul e tumo rinduction

. Nevertheless , i n a specialize d initiation-promotio n stud y wit h unleade d gasolin e an d TMP , where th e authors quantifie d foc i o fCPN , som e adenoma s were describe d a s arisin g withi n foc i o fCP N (Shor t e t al. , 1989b)

. E. Evidence

Concerning

Progression

from Nephrotoxicity

to Renal Neoplasia Fo rth e eigh t selecte d CIG Acarcinogen s examine d i n thi s report , ther e was a n overal l patter n indicativ e o fdose-relate d increase s i n th e incidence s oftoxi cnephropathy , hyperplasia , an d rena l tubul e tumors i n mal e rats

. Fo r tw o CIGA , unleade d gasolin e an d TMP , dose-relate d increase s i n rena l tubul e proliferatio n were sustaine d throughou t chroni cadministration

. I t i s believe d that th e likelihoo d ofproducin g a cancerou s cel l i s increased , not onl yi fthere i s a probabilityo fa geneti ctransition , bu t als o i fth e rat e o fcel l replicatio n i s increase d (Cohe n an d Ellwein , 1990 , Dea l et al.

. 1989)

. Thus , a sustaine d stat e o fcel l turnove ri n th e targe t cel l populatio n a s a mecha nisti clin k betwee n a^- g nephropath yan d rena l neoplasi a shoul d b e considere d a plausible , bu t unproven , explanatio n o fth e observe d results

. Th e hyperplasti ctubule s an d adenoma s produce d b yCIG Acarcino gen s appea rt o aris e fro m th e cortex , whic h include s th e P 2 segmen t o fth e proxima l tubule , th e mai n sit e o fcellula rinjur

yi n ot

2u 

### - g nephropathy , providin g furthe rsuppor t fo rthei rlinkage

. Furthermore , studie s that examine d cel l regeneratio n i n th e differen t segment s o fth e mal e rat kidne y have show n a n increas e i n cel l replicatio n rate s specificall yi n th e histologi -

### call ydamage d P 2 segment s afte radministratio n o ftetrachloroethylen e o r pentachloroethan e (Goldsworth yet al.

. 1988a) o rTM P o runleade d gaso lin e (Shor t et al.

. 1987

. 1989a)

. Unde rth e sam e conditions , tubul e cel l replicatio n i n femal e rat s di d no t diffe rfro m control s i n an yo fthes e studies , no ri n rat s ofbot h sexe s treate d wit h a non-CIGA

. trichloroethylen e (Goldsworth yet al. , 1988a)

. Recen t studie s o

fth e promotio n potentia l o fd-limonene , TMP

. an d gasolin e als o provid e convincin g evidence t o suppor t a linkag e betwee n OL- g nephropath yan d rena l tubul e neoplasia

. Dietric h an d Swenberg (T99lb ) demonstrate d that d-limonen e promote d rena l tubul e tumor s i n mal e F34 4 rats , a n anima l that produce s o_-g . I n addition , ther e was a 5 - fol d increas e o fP2-labelin g inde xi n th e F34 4 rat s treate d wit h d-limonene

. Incontrast , n o respons e wa s recorde d fo rproliferation , hyperplasia , o rrena l tubul e adenoma s i n th e NB R rat , a n ot^-g-deficien t anima l whic h doe s no t develo p th e characteristi cnephropathy

. Thes e result s substantiat e thos e o fa n earlie rstud ywher e dose-relate d increase s i n atypica l cel l foc i wer e observe d i n mal e rat s promote d wit h unleade d gasolin e o rTM P fo r2 4 o r6 0 week s (Shor t etal.

. 1989b)

. I n that study , ther e wa s a significan t linea rtren d i n incidenc e o frena l tubul e tumor s i n th e mal e ra t promote d wit h unleade d gasolin e fo r2 4 weeks

. I n contrast , non e ofthes e change s wa s observe d i n similarl ytreate d femal e rats

. Finally , th e nephrotoxicitysee n i n mal e rat s i n th e selecte d 2-yea r bioassays ofrena l tubul e carcinogen s wa s characteristi co ftha t propose d t o resul t fro m cel l damag e cause d bya_- g accumulation

. I n contrast , wheneve rnephrotoxicitywa s observe d i n femal e rats , o rmic e ofeithe rsex , i.e.

. fo rhexachloroethane

. tetrachloroethylene

. an d 1,4-DCB , th e lesion s were no t characteristi co fCIG Aan d probabl ywer e a respons e cause d b y a n independen t mechanism

.

7 2 

## Par t 3 - Evaluatio n o fth e Hypothesi s

XI . Summar

yo fth e Evidenc e o n th e Rena l Effect s o fCIG A Severa l line s o fevidenc e establis h a n associatio n betwee n exposure o f th e mal e ra

t t o chemical s tha t induc e o^- g accumulatio n (CIGA) an d nephrotoxicity , an d strongl ysupport a n associatio n betwee n thi s nephrotoxicityan d rena l tubul e tumors

. A. Association

Between

Alphas-globulin

Accumulation

and Nephrotoxicity Th e informatio n that support s a n associatio n betwee n OL-g accumula tio n an d mal e rat-specifi crena l toxicityfollowin g CIG Aadministratio n i s summarize d below

. Althoug h hyalin e drople t accumulatio n pe rs e i s not necessaril y diagnosti co

fa CIG Aunti l prove n t o represen t EL- g accumulation , 3 4 organi ccompound s includin g fuels , solvents , an d othe r chemical s (liste d i n Tabl e A-1) , examine d i n thi s report , hav e bee n show n t o induc e a n excessiv e accumulatio n o fhyalin e droplet s i n th e rena l proxima l tubul e epitheliu m ofmal e rats

. I n contrast , where tested , mic e an d femal e rat s showe d n o evidenc e ofhyalin e drople t accumulatio n fro m chemica l treatment

. There i s convincin g evidenc e tha t th e excessiv e accumulatio n ofhyalin e droplet s i s followe d sequentiall yb ytubul e epithelia l cel l necrosis , granula rcas t formation , an d othe raspects o f a^- g nephropath yi n th e mal e rat

. Fiv e o fth e 3 4 hyaline-drople t inducers were teste d i n specie s othe rtha n th e mous e o rth e rat , althoug h possibl yno t a s rigorously

. Characteristi clesion s wer e observe d i n th e mal e rat kidne yfo rthes e fiv e substances , bu t ther e wa s n o apparen t nephrotoxi crespons e i n th e femal e ra t o ran yothe rspecie s tested , whic h include d mice (al l 5 substances) , hamsters (je t fuels) , guine a pig s (decalin) , dog s (je t fuels , decalin , d-limonene , an d methy l isobuty l ketone) , an d monkeys (methy l isobuty l keton e an d gasoline)

. Th e increas e i n hyalin e droplets , tubul e dilatio n cause d b y granula rcast formation , tubul e cel l proliferation , an d medullar y mineralizatio n i s dose-dependen t a s show n b yresearc h studie s conducte d t o dat e wit h fou rmode l CIG A(decalin , d-limonene , unleade d gasoline , an d TMP)

. I n general , th e chroni cadministratio n o fCIG At o mal e rats an d th e ensuin g nephrotoxicityenhance d th e age-relate d rena l degenerative proces s b yexacerbatin g spontaneou s CPN

. 7 3Specialize d studie s involvin g rat s o fvaryin g age , castrate d o r

estrogen-treate d rats , th e NB R strain , an d ot^-g-treate d femal e rat s hav e show n tha t developmen t o fth e earl yfeature s o f a^- g nephropath yi s dependen t o n th e presenc e o fct^- g forme d i n th e liver

. Fo rthre e o fth e eigh t mode l carcinogen s (hexachloroethane , tetrachloroethylene , an d 1,4-DCB) , rena l toxicit ywa s observe d i n chroni cstudie s o ffemal e rat s o rmice , bu t th e rena l toxicit y appeare d t o b e les s sever e o rqualitativel ydifferent , no t involvin g th e sam e spectnj m o fdiscret e lesion s associate d wit h a^- g nephropathy

. •CIG Abin d reversibl yt o a^- g a s a targe t molecule , an d th e rena l accumulatio n o fa^- g an d hyalin e drople t formatio n ma yb e explaine d b ychemicall yinduce d impairmen t o fa^- g catabolis m afte rreabsorptio n o fth e comple xb yth e proxima l tubule

. •TMPOH , th e activ e metabolit e o fTM P ma yb e abl e t o for m i n vitr o complexe s wit h retinol-bindin g protei n an d a,-aci d glycoprotein , member s o fth e lipocali n superfamil yfoun d i n humans

. I n vh/odat a o n retino l an d a^-g , however , demonstrat e tha t suc h a n associatio n doe s no t necessaril ylea d t o ot^- g accumulatio n o rhyalin e drople t formation

. B. Association

Between

Nephrotoxicity

and Renal Cancer Base d o n informatio n fro m th e roden t bioassay s examine d i n thi s repor t an d additiona l ke ydata , feature s o frena l tumor s occurrin g subsequen t t o th e developmen t o fnephropath yi n th e mal e ra t ca n b e identified

. •Th e eigh t mode l carcinogen s produce d hyperplasia , adenomas , an d adenocarcinoma s i n th e rena l tubul e o fth e mal e rat

. •Al l eigh t tha t produce d rena l tumor s i n mal e rat s als o produce d nephrotoxicit yi n mal e rats

. Specifically , th e nephrotoxicit ytha t precede d rena l tumo r formatio n i n mal e rat s wa s characteristi co fth e for m associate d w <l h a^- g an d distinguishabl e fro m othe rform s o ftoxicit y associate d wit h non-CIG Arena l toxicants

. Th e incidenc e o frena l tumor s produce d i n th e mal e ra t b yth e eigh t mode l carcinogen s wa s relativel ylow

. Thes e tumor s wer e morphologicall yindistinguishabl e fro m othe rchemicall y induce d rena l tubul e neoplasi a an d rena l tubul e neoplasi a tha t occur s rarely , bu t spontaneously , i n mal e an d femal e rats

. Th e rena l tumor s produce d b yth e eigh t mode l carcinogen s occurre d late , usuall ybein g foun d a t th e tim e o fsacrifice , metastasize d rarely , an d wer e no t life-threatening

. Fo rd-limonene , th e on e CIG Aexamine d i n a n initiation promotio n stud ycomparin g mal e rat s o fth e NB R strai n wit h a conventiona l strain , a^- g accumulatio n wa s necessaryfo r promotio n o fmal e ra t rena l tubul e tumor s initiate d b yEHEN

. CIG Aappea rt o b e nongenotoxi co ronl ymarginall ys o and , therefore , ma yno t depen d o n direc t geneti cinjur ya s th e mechanis m fo rtumo rinduction

. Trichloroethylene , a compoun d structurall ysimila rt o hexachloroethan e an d tetrachloroethylen e produce d rena l tumor s apparentl yb ymechanisms , suc h a s covalen t bindin g t o DNA , whic h d o no t appea rapplicabl e t o th e CIG Ahypothesis

. C. Information

Reducing

Confidence

In the Conclusion

that the Alphas-globulin

Response

Is Specific to the Male Rat Althoug h th e evidenc e availabl e t o dat e support s th e hypothesize d associatio n betwee n a^- g accumulatio n an d rena l tubul e tumor s i n th e mal e rat , confidenc e i n thi s assertio n woul d b e improve d i fth e sam e result s wer e foun d i n a n expande d database

. I n addition , th e paucit yo fdat a o n th e lipocali n superfamily , i n general , leave s severa l question s unanswere d regardin g th e specificit yo fth e respons e t o th e mal e rat

. Pathologica l accumulatio n ofhyalin e droplet s i s a reactio n t o excessiv e protei n loa d no t exclusivel yrelate d t o a 2 - g accumulation

. Althoug h ther e ar e 3 4 hyalin e droplet-inducin g compound s identifie d i n Tabl e A- 1 o fthi s report , th e accumulatin g protei n responsibl e fo rhyalin e drople t formatio n ha s bee n identifie d fo ronl y1 7 o fthes e compounds

. Dat a sufficien t t o demonstrat e interdependenc e o fth e lesion s i n th e propose d pathologica l sequenc e fro m hyalin e drople t accumulatio n t o chroni ctoxicit yexis t fo ronl ya fe wsubstances

. Dat a t o defin e dose-respons e relationship s fo rtubul e cel l necrosi s an d it s associatio n wit h cel l proliferatio n ar e eve n mor e limited , a s i s dose-relate d informatio n o n increase d cel l proliferatio n rate s ove rchroni cexposur e periods

. Th e mechanis m whereb ya 2u - g accumulatio n lead s t o cel l deat h ha s no t bee n established

. Hexachloroethane , tetrachloroethylene

. an d 1,4-DC B produce d rena l toxicit yi n femal e rat s o rmic e indicatin g tha t som e CIG A ma yhav e additiona l effect s o n roden t kidne yno t limite d t o th e oL-g-induce d sequenc e o flesions

. Informatio n o n a possibl e associatio n betwee n rena l cel l tumor s an d CIG Aexposur e i n human s i s inconclusiv e sinc e exposure s i n th e reviewe d epidemiologi cstudie s hav e bee n t o bot h CIG A an d non-CIG Acompounds

. Informatio n o n th e i n viv o bindin g o l CIG Awit h othe rlipocalin s i n th e rx, u - g superfamil yofprotein s suggests , bu t doe s no t conclusivel ydemonstrate , tha t toxicit yi n human s coul d no t occu rvi a thi s mechanism

. Althoug h ther e ar e majo rquantitativ e an d qualitativ e difference s betwee n mal e rat s an d human s i n th e amount s o fprotei n excrete d i n urine , littl e i s know n concernin g th e relativ e quantitie s oflow-molecular-weigh t protein s tha t ar e normall yfiltere d b y th e huma n glomerulu s an d reabsorbe d b yth e rena l tubule s fo r catabolism

.. i n e screntiii c dat a summarize d abov e wer e use d t o dra wconclusion s concernin g th e rol e o

fOL- g accumulatio n an d hyalin e drople t formatio n i n producin g mal e rat-specifi c nephropath y an d rena l tubul e neoplasia , an d t o determin e th e relevanc e o fthi s informatio n t o assessin g huma n risk. XII . Conclusion s Th e availabl e informatio n o n CIG A associate d rena l tubul e carcinogenesi s i n th e mal e ra t ca n b e describe d by a suggeste d sequenc e o fcritica l cellula ran d molecula revents . Accordin g t o thi s description , th e reactio n o

fa lipophili c compoun d wit h th e low-molecular-weigh t protein , ot^-g , appear s t o lea d to th e formatio n o fa complexthat i s mor e resistan t t o lysosoma l degradatio n tha n th e unreacte d protein . Thi s results i n a shif t i n balanc e betwee n reabsorptio n an d hydrolysi s leadin g to a n abnorma l accumulatio n o

fth e protei n i n th e P 2 segmen t o fth e rena l tubul e o

fmal e rats . Ifexposure cease s afte ra shor t tim e period , recover y i s complete . Continue d exposure , however , result s i n a nephrotoxi c respons e tha t i s les s readil y reversibl e an d a sustaine d increas e i n cel l turnover , enhancin g th e chanc e that molecula ralteration s i n DN A occurrin g i n th e kidney may b e replicate d rathe rtha n repaired . Becaus e ther e ar e substantia l dat a gaps , especiall y wit h regar d t o th e expecte d respons e i n human s an d th e critica l linkage s betwee n single-cel l necrosi s an d increase d cel l turnover , an d tubul e hyperplasi a an d rena l tubul e cancer , th e 0L u -g syndrom e shoul d b e considere d a satisfactor y workin g hypothesi s bu t no t a prove n mechanis m o factio n to describ e rena l tubul e cance ri n mal e rat s expose d t o CIGA . A s such , i t provide s a n empirica l descriptio n o fa serie s o

fobserve d event s i n laborator y animal s whic h coul d b e modifie d o rexpande d upo n a s additiona l informatio n become s available . Despit e thes e limitation s an d th e fact that a^- g accumulatio n als o exacerbate s CPN , chemicall y induce d OL-g-associale d nephropath y i n th e mal e ra t ca n b e distinguishe d histopathologicall y fro m othe rchemicall y induce d nephrotoxicitie s an d als o fro m CPN . Excessive hyalin e droplet formatio n i s th e earliest morphologi c manifestatio n an d a n importan t char acteristic , althoug h a chemica l can b e describe d a s a CIG A wit h certainty onl y whe n ther e i s a positiv e identificatio n ofOL- g i n th e hyalin e droplets . Othe robservabl e characteristic s indicative o fpossibl e CIGA-induce d nephrotoxicity includ e single-cel l necrosi s o fth e tubul e epithelium , granula r cast s a t th e junctio n o fth e inne ran d oute rstripe s o fth e oute rmedull a cause d b y sloughin g o fnecroti c cells , mitoti c figure s indicativ e o fregenera tio n o rincrease d cel l turnover , an d ofte n medullar y mineralization . Th e hepati c synthesi s o fth e lipocalin , O L -g , i s no t know n to occu r normall y i n an y specie s othe rtha n th e mal e rat . Alpha2u -globulin-induce d nephropath y i s als o a distinc t entity specifi c t o th e mal e ra t amon g th e laborator y specie s an d genders teste d t o date . Th e characteristi c nephropath y ha s bee n foun d onl y whe n a^- g forme d i n th e live ri s present . Thus , femal e rat s d o no

t develo p hyalin e droplet s whe n expose d t o CIG A unles s they 7 6 hav e bee n administere d OL . - g isolate d fro m mal e ra t urine . NB R rat s whic h d o no t posses s th e mRN A fo rlive ra 2u -g , an d castrate d mal e rats , als o respon d differentl y fro m conventiona l mal e rats . O fth e othe rspecies , th e mous e i s th e mos

t thoroughl y tested . Althoug h th e mous e produce s larg e amount s o

fa structurall y simila rlipocalin , MUP , thi s protei n i s no t know n to bin d wit h CIGA ; i

t i s not reabsorbe d fro m th e urine ; an d th e mous e doe s not develo p kidne

y tumors o rth e characteristi c nephropath y see n i n mal e rat s Limite d testin g i n dogs , hamsters , guine a pig s an d monkey s ha s no t show n hyalin e drople

t accumulatio n o rnephropath y i n thes e species , furthe r suggestin g that th e ot^- g syndrom e occur s specificall y i n th e mal e rat . Wit h regar d t o th e potentia l fo ra chemica l t o produc e rena l tubul e neoplasi a i n th e mal e rat , ther e ar e commo n characteristic s amon g th e substance s evaluate d i n thi s report . First , thes e compound s (an d thei r CIGA-bindin g metabolites ) posses s littl e o rn o mutageni c activity i n stan dar d batterie s o

ftests , the y ar e lipophile s an d no t electrophili c substances , an d they d o no

t appea rt o bin d covalentl y t o DNA . Second , th e nephrotoxi c respons e characteristi c o fCIG A alway s precede d rena l tumo rformatio n i n th e mal e rat , a findin g not characteristi c o fclassica l rena l carcinogens . Third , fo ral l eigh t mode l compound s examine d i n thi s report , additiona l sexes/species/strain s wer e tested , an d th e increase d incidenc e o frena l tumor s wa s foun d onl y i n th e mal e rat . Th e manne ri n whic h th e huma n mal e respond s t o CIGA ha s no t bee n teste d directl y althoug h ther e ar e huma n protein s that , lik e ot^-g , ar e member s o

fth e lipocali n superfamily . Huma n urin e als o contain s smal l amount s o

fa sex-linke d urinar y protein . Epidemiologi c studie s hav e focuse d o n glomerulonephriti s o rrena l cance ran d organi c chemica l expo sure , i n general , an d no t o n rena l tubul e damag e an d CIG A exposure , an d the y d o no

t yiel d result s usefu l fo rtestin g th e hypothesize d mechanis m i n humans . Protei n overloa d can resul t i n formatio n o fhyalin e droplet s i n huma n kidneys , althoug h ther e i s n o evidenc e that thi s respons e ha s occurre d fro m lipocali n accumulation . Whil e i t i s not possibl e t o resolv e th e issu e o fho wth e huma n rena l tubul e respond s to CIG A exposur e fro m th e availabl e data , th e uniquenes s o fth e mal e rat respons e amon g th e teste d laborator

y specie s an d th e hig h dose s neede d t o produc e a n effect , eve n i n th e mal e rat , suggest that thi s reactio n woul d not occu ri n humans , especiall y unde rtypica l condition s o fexposure . Severa l factors complicat e th e analysi s o fdat a o n th e rena l effect s o f CIGA . Fo rth e compound s examine d t o date , no t al l o fth e administere d substanc e ha s bee n foun d t o bin d t o OL-g . Thu s additiona l CIGA/CIG A metabolite s potentiall y exis t i n th e kidney alon g wit h th e OL-g-boun d material . Th e possibilit y that thes e othe rmoietie s ar e toxi c t o th e kidney need s t o b e take n int o account . Fo rexample , tetrachloroethylene , i n additio n t o showin g a^- g nephropathy , display s evidenc e o frena l toxicit

y typica l o fchlorinate d hydrocarbons . Thi s exampl e demonstrate s howothe r mechanism s ma y play som e rol e i n th e observe d results . A t present , ther e i s insufficien t informatio n o n CIGA an d thei rmetabo lite s to confident; /

predict activit

y o n th e basi s o

fstructura l analogy . Recen t 7 7

### researc h o n structura l correlation s suggests tha t th e presence o

fa n electro negativ e ato m fo rhydroge n bonding , lipophilicity , an d steri cvolum e are importan t considerations

. Conformationa l change s o rothe rstructura l alteration s t o th e protei n ma yals o b e necessar

ysinc e bindin g ofth e compoun d i n th e protei n pocket , alone , appear s t o b e a n insufficien t conditio n t o caus e reduce d digestibilityo fth e protein

. Evidence o

fdose-responsivenes s betwee n CIG Aadministratio n an d th e degre e o

fhyalin e drople t o ra^- g formatio n ha s bee n demonstrate d i n severa l studies

. However , thes e finding s ar e frequentl ybase d o n subjectiv e histopathologica l criteria , limitin g thei rusefulnes s fo rmakin g quantitativ e judgments abou t th e relativ e hazard potentia l o fdifferen t chemicals

. I t i s als o importan t t o recogniz e that fo rvariou s reason s (e.g. , dose s administere d to o low , animal s kille d befor e th e latenc

yperio d o

fthes e slow growin g tumors i s attained , numbe ro fspecimen s an d histologica l section s insufficient , competin g toxicityi n kidneyo rothe rorgans) , th e entir e patho logica l sequence culminatin g i n rena l tubul e neoplasi a ma ynot b e demon strate d i n al l case s o fCIG Aadministration

. Thus , not al l CIG Awoul d b e expecte d t o demonstrat e rena l tubul e neoplasi a i n th e mal e rat i n a 2-yea r anima l bioassay

. Suc h a findin g woul d no t negat e th e applicabilityofth e hypothesize d CIG Asyndrom e t o th e evaluatio n o fnephropath ydata

. Base d o n th e cance rbioassays an d othe rresearc h studie s ofCIGA , a n increase d proliferativ e respons e cause d b ychemicall yinduce d cytotoxicity appear s t o playa rol e i n th e developmen t o frena l tubul e tumor s see n exclusivel yi n mal e rats

. Th e mal e ra t specificit yo fth e respons e t o CIG A administratio n i s emphasize d bynegative finding s i n mic e an d femal e rats

. Thes e conclusion s ca n probabl yb e extende d t o analysi s o fhuma n hazar d potential , especiall ywheneve rhuma n exposur e t o CIG Ai s no t excessivel y hig h to rsustaine d period s o ftime , whe n short-ter m test s fo rgenotoxicityo f th e compoun d are negative , whe n th e nephrotoxi crespons e an d increase d cel l turnove rcharacteristi co fCIG Ahav

e bee n demonstrate d i n th e mal e rat , an d othe rspecies/se xcombination s wer e teste d bu t rena l tubul e tumor s were observe d onl yi n mal e rats

. XIII . Researc h Need s Certai n studies , suggeste d t o fil l ke ydat a gaps , are liste d below

. Ther e has bee n n o attemp t t o outlin e al l th e possibl e avenue s fo rresearc h o t i CIG A an d o n lipocalins , sinc e a vas t arra yo fusefu l experiment s coul d b e envisioned

. Instead , recommende d studie s woul d greatl yimprov e th e databas e o n thes e chemicals , provid e neede d informatio n t o answe r question s o

fhuma n relevance , an d set u p a framewor k fo rimprovin g th e testin g o fchemical s tha t are potentiall ymal e ra t rena l tubul e tumorigens

. Thes e researc h need s are liste d as follows : Exten d studie s i n humans , whereve rpossible , t o determin e directl yth e effect s o

fhydrocarbo n an d solven t exposure , focusin g o n specifi cjob s wit h relativel ypure CIG Aexposure . An yhuma n rena l patholog yfoun d shoul d b e compare d wit h oc^- g nephropath yi n th e mal e rat

. an d urin e shoul d b e examine d fo rth e presence o

fcell s an d cast s sinc e thi s noninvasive techniqu e i s readil yapplie d t o humans

. Examin e huma n subpopulatton s tha t excret e abnorma l amounts o flow-molecular-weigh t protei n i n th e urin e t o determin e ifthe y are a t ris k o frena l diseas e o rrena l cel l cancer . Examin e additiona l activ e CIG Ametabolite s fo rbindin g t o lipocalins

. suc h a s retinol-bindin g protein , a,-aci d glycoprotein , an d urin e protei n 1

. I fthere i s binding , determin e i t th e protei n complexha s a slowe rdegradatio n rate

. Thoroughl ycharacteriz e an yprotei n drople t nephrotoxicity observe d a s th e resul t ofadministerin g know n CIG A(e.g.

. d-limonene

. TMP ) t o additiona l specie s (e.g.

. dog , hamster , rabbit , guine a pig , an d especiall ynonhuma n primate)

. Develo p a standardize d short-ter m protoco l (e.g. , i n th e 2-wee k subacut e study) that wil l detec t an yabnorma l accumulatio n o f hyalin e droplet s i n th e mal e rat kidne ybefore suspecte d CIG A are place d o n chroni cstudy

. (I fhyalin e drople t accumulatio n i s found , thi s informatio n shoul d b e take n int o accoun t i n designin g th e bioassay. ) Furthe rcharacteriz e th e respons e o fth e NB R rat , whic h doe s no t appea rt o synthesiz e a^-g

. t o CIG Aan d t o classica l rena l carcinogens

. Thes e studie s shoul d verify , i n a two-yea rchroni c bioassay , that th e NB R ra t kidne yi s responsiv e t o classica l rena l carcinogen s alread yteste d i n othe rstrains , an d the y shoul d evaluat e th e suitabilityofthi s strai n o fra t a s a tes t species

. I fth e NB R rat meets thes e tw o criteria , th e possibility ofemployin g a separat e tes t group , consistin g o fmal e NB R rats , shoul d b e considere d fo rconventiona l bioassays wheneve r excessiv e hyalin e drople t formatio n ha s occurre d i n shorter ter m tests

. Conduc t serial-sacrific e studie s o fCIG Aan d non-CIG Arena l carcinogen s t o determin e i fa distinctl ydifferen t progressio n fro m OL- g nephropath yt o tumo rformatio n ca n b e see n fo r CIGATstudie s shoul d involv e chroni cexposures , examin e th e histogenesi s o

fth e rena l tubul e tumors , an d includ e "stop " experiment s an d time-dependen t appearanc e o ftumo rmarkers

. Perfor m dose-respons e studie s designe d t o quantitat e th e relationshi p betwee n increase d hyalin e droplets an d cel l necrosi s an d betwee n cel l necrosi s an d cel l regeneration

. I n addition , explore th e possibilit

yo fadditiona l step s i n th e progressio n that migh t furthe rdefin e th e expressio n ofcance ri n th e mal e rat an d th e caus e o

fcel l death

. Conduc t metabolis m an d dispositio n studie s o fCIG Ai n othe r species , compare d wit h mal e rats , t o determin e th e causativ e chemica l lo rth e nephropathy , an d t o clarif ysite s of biotransformatio n an d depositio n an d fat e o fthes e compounds

.

7 8 7 9Additiona l work , no t a s critica l a s th e above , bu t whic h woul d als o assist i n understandin g thi s diseas e process , include s th e following : Identif yth e accumulatin g materia l containe d i n hyalin e droplet s o fproxima l tubules fo rchemical s that are apparent , bu t unverifie d CIGA , an d conduc

t 2-yea rbioassay s fo rdecali n an d TMP . Perfor m additiona l i n vitro assay s usin g roden t kidne yextract s t o more specificall ydetermin e mutageni c potentia l o fCIGA (o r activ e metabolites) . Conduct studie s o n th e genesi s o fCP N an d its relationshi p t o a^- g nephropath yan d examin e th e possi>te rol e o fCP N a s a cocarcinogeni c facto rfo rrena l tumo rinduction . Obtai n more informatio n o n th e rena l catabolis m o fOL- g an d th e rat e an d efficiencyo fprotease-mediate d hydrolysi s i n contro l an d CIGA-treate d rats . Stud yth e bindin g relationship s betwee n CIG A an d OL- g (e.g . affinity , concentratio n ranges , bindin g effectors ) an d determin e th e site at whic h bindin g ofCIG A t o OL- g occurs (e.g. , Ih/er , plasma , o rurine) t o investigat e th e hypothesi s that th e protein CIG A comple

xi s onl yforme d at hig h concentration s o fth e chemical . Determin e th e reason s wh yth e amoun t oftow-molecularweigh t protei n i n huma n urin e i s muc h les s tha n i t i s i n mal e rats .

8 0 Par t 4 . Scienc e Polic y

XIV . Backgroun d an d Introductio n A n Increase d incidenc e o fneoplasm s i n laborator yanimal s adminis tere d test chemical s i s customaril yviewe d b yscientist s a s a n Indicatio n o f carcinogenicit

yi n animal s an d a s som e signa l that human s ma yb e similarl y affected . Fro m thi s lin e ofreasoning , EP A generall ypresume s tha t anima l tumo rfinding s Indicat e ther e ma yb e a cance rhazar d t o humans , althoug h a fina l Judgment a s t o huma n carcinogeni c potentia l ca n b e mad e onl yI n relatio n t o al l othe rrelevan t Information . Recen t studie s suggest , however , tha t tumors produce d i n th a tubul e ofth e mal e ra t kidne yfollowin g th e accumulatio n o

falphas-globuli n (OL-g ) migh t involve a proces s tha t occur s onl yI n th e mal a rat . Becaus e ofth e implication s t o cance rris k assessment , th e Ris k

Assessmen t Foru m (RAF ) establishe d a Technica l Pane l t o examin e th e availabl e informatio n o n a^- g accumulatio n i n th e kidney , associate d rena l disease , an d kidneycancer . Th e scientifi c dat a supportin g th e Technica l Panel' s conclusion s regardin g th e ot^- g sequenc e o flesions * ar e covere d i n dept h i n th e precedin g section s (Part s 1 throug h 3 ) ofthi s document . Part 4 provide s guidanc e t o EP A risk assessor s regardin g evaluatio n of mal e rat kidneytumor s an d present s RA F conclusion s regardin g potentia l huma n hazar d an d risk fo ra specia l subset ofthes e tumors , tha t is , rena l tubul e tumors i n th e mal e rat resultin g fro m chemicall y induce d cc- g accumulation . Criteri a fo rdemonstratlngthi s relationshi p ar e se t fort h belo w fo rus e an d discussio n I n al l EP A assessment s i n whic h dat a o n rena l tubul e tumor s I n th e mal e ra t ar e use d t o asses s huma n risk . XV . Basl e fo rScienc e Polic yo n Mal e Ra t Kidne yTumor s Th e informatio n tha t follows highlight s critica l dat a an d outline s Inferen tia l bridge s use d t o selec t th e most plausibl e explanatio n fo rth e Informatio n availabl e o n mal e ra

t kidne ytumors . A . Low-Molecular-Welgh t Protein s I n th e Ra t I n ra t kidneys , a s i n thos e ofothe rmammals , naturall yoccurrin g lowmolecular-weigh t protein s are transferre d fro m th e plasm a int o th e urin e b y glomerula rfiltration . Th e protein s ar e the n partiall yreabsorbe d fro m th e urin e Into th e rena l tubul e o fth e kidneywher e the yar e eventuall ybroke n dow n b ycatabolis m (se e sectio n lll-A) . On e o fthes e low-molecular-weigh t proteins , o^- g produce d byth e live runde rth e stimulu s o ftestosterone ,

* I n thi s report , lesio n i s a morphologica l alteration , du e t o disease . 8 1reaches ver

yhig h level s i n th e plasm a an d urin e o fyoun g adul t mal e rats , graduallydeclinin g wit h age . Alphas-globuli n i s regarde d a s a membe ro fa larg e superfamU y o f protein s thought t o b e carrier s o flipophili c molecule s (se e sectio n lll-D) . Som e o fthes e proteins , e.g. . retinol-bindin g protei n an d a,-aci d glycopro tein , are foun d i n man yspecies , includin g humans . Others , lic e ot^-g , ar e foun d i n specifi c species . Th e onl y membe ro fth e superfamil y wit h a clearl y defined physiologica l rol e i s retinol-bindin g protein , th e carrie rprotei n fo r vitami n A Althoug h thes e low-molecular-weigh t protein s are believe d t o hav e simila rthree-dimensiona l structures , th e alignmen t o famin o aci d residue s betwee n an ypai ro

fprotein s i n th e superfamil yi s small , roughl y2 0 percent . Th e exceptio n i s oc^- g an d mous e majo rurinar yprotein(s ) (MUP ) whic h are approximatel y9 0 percen t homologous . Alphas-globuli n derive d fro m hepati c synthesi s i s no t know n t o occu ri n th e femal e ra

t o ran yothe rspecies , includin g humans . Althoug h simila r forms o fo_- g ar e synthesize d a t nonhepati c site s i n femal e rat s an d i n th e mal e NC I Blac k

Reite r(NBR ) rat , a strai n whos e male s lac k hepati c synthesi s o l OL-g , non e o fthes e othe rform s o

fa^- g no rMU P accumulate s I n th e rena l tubul e followin g administratio n o fth e compound s discusse d i n Part s 1 throug h 3 . B. Progression

from Chemically

Induced

Alphas-globulin Accumulation

to Nephropathy

and Neoplasia 1. Overview Th e informatio n availabl e provide s a plausible , althoug h probabl y incomplete , picture o

fa sequenc e o

fevent s occurrin g i n th e mal e rat kidney followin g chemica l administration . Thi s sequenc e ca n b e portraye d o n a cellula ran d molecula rlevel . Initially , th e test chemica l appear s t o bin d reversibr y t o ot^-g , seemingl yformin g a comple xmore resistan t t o lysosoma l degradatio n tha n th e unreacte d protei n itsel f(se e sectio n lll-H) . Thi s shifts th e balance betwee n reabsorptio n an d catabolis m an d appear s t o resul t i n accumulatio n o

fth e protei n complexi n a specifi c are a o fth e rena l tubule , th e P 2 segment . Continue d compoun d administratio n result s i n a cytotoxi c respons e fro m th e sustaine d protei n overloa d t o th e rena l tubule , causin g single-cel l necrosi s o

fcell s linin g th e surfac e o fth e tubul e an d othe rkidney pathology . Th e dea d cell s are replace d b ycel l division . As th e cycl e o fcel l deat h an d cel l replacemen t continues , wit h tim e tubul e hyperplasi a (in crease i n numbe ro

fcells ) an d neoplasi a ma yoccur . It i s presumed , bu t certainl yno

t proven , that continue d cel l proliferatio n plays a rol e i n th e neoplasti c process . Morphologically , th e sequenc e o fevent s begin s wit h a n increas e i n th e numbe ran d siz e o fhyalin e droplets 6 containin g ot^-g . Th e nex t

' Spherica l inclusion s i n th e cytoplas m tha t ma ycontai n variou s protein s (se e sectio n III B)

. 

### 8 2 characteristi c lesion , single-cel l necrosi s i n th e rena l tubule , mayno t b e see n bu t can b e confirme d b yobservatio n o

fexfoliate d degenerat e cell s i n th e tubul e lumen 7 an d granula rcasts. ' Enhance d cel l replicatio n i n respons e to cel l deat h can b e see n a s increase d cel l divisio n o rdemonstrate d b y labelin g technique s that measur e increase d DN A synthesis . I n chroni c laborator yanima l bioassays , tubul e hyperplasia , linea rmineralizatio n i n th e rena l papill a (possibl yrepresentin g remnant s o fdebri s fro m disintegratin g granula rcasts) , an d rena l tubul e tumors ar e observed . 2. Specificity

of the sequence

to the male rat Consisten t result s fro m hypothesis-testin g experiment s conducte d ove r th e last decad e i n variou s laboratorie s establis h th e associatio n betwee n th e accumulatio n o

fabnorma l amounts o

fa 2u - g i n th e P 2 segmen t o fth e rena l tubule s an d a specifi c for m o fkidne ydisease , an d the ysuppor t a n associa tio n betwee n thi s nephropathi c respons e an d rena l tubul e tumors . Specifi cally , th e mal e rat respond s t o administratio n o fot^- g inducer s wit h a characteristi c nephropathy . Th e severityo fthi s kidne ydiseas e i s dose dependent , no

t onl ywit h respect t o th e amoun t o fcompoun d administered , bu t als o wit h respect t o th e concentratio n o fOL^- g i n th e kidney . Thi s alpha. - gtobuli n nephropath ydiffers sufficientl yfromchroni c progressive nephropath y (se e sectio n IV-E ) commonl yfoun d spontaneousl yi n agin g mal e rat s s o that th e two effect s can b e distinguished . I n contrast , mic e an d femal e rat s administere d a 2u -g-inducer s unde rth e sam e condition s a s mal e rat s di d no t develo p lesk>ns u characteristi c o fot^- g nephropathy . Hyalin e droplet s i n th e proxima l tubul e o funtreate d mal e rat s contai n CL^g , especiall yi n youn g adults . Hyalin e droplet s ar e substantiall yreduce d i n castrate d mal e rats , furthe rindictin g th e dependenc e o fthi s phenomeno n o n mal e hormon e levels . I n femal e rats o fan yage . a n observatio n o fprotei n droplet s i s rar e an d OL^- g i s no t involved . Specialize d studie s involvin g hormon e manipulatio n hav e show n that th e developmen t o

fth e earl y feature s o l a^- g nephropath yi s dependen t o n th e presenc e o

fth e hepati c for m o

fa 2u - g (1 ) Hyalin e droplet o rOL- g accumulatio n doe s no t occu rwhe n oL-g - inducer s ar e administeredt o immatur e o rol d mal e rat s tha t produc e littl e ot^- g i n th e liver . (2 ) Hyalin e droplet accumulatio n i s observe d fro m administratio n o fa 2u -g-inducer s eve n i n castrate d rats , bu t th e severit yo fth e effec t i s diminished . (3 ) Estroge n administratio n t o mal e rats reduce s th e severityo fO L - g nephropathy . (4 ) Femal e rat s administere d a n a 2u -g-induce ralon g wit h a^- g purifie d fro m mal e rat urin e clearl yshowe d hyalin e droplet formation , cL, u - g accumulatio n i n th e kidney , an d som e nephropath yeve n thoug h contro l femal e rats showe d n o measurabl e effects .

7 Cell s insid e th e tubule , whic h sho wcytoplasmi cdeterioration , arisin g fro m th e damage d P 2 segmen t (se e sectio n I V A )

* Th e granula rcast s ar e compose d o fsloughe d cel l debri s fro m th e dea d cells

. The y accumulat e a t th e junctio n betwee n th e P 3 segmen t o fth e proxima l tubul e an d th e descendin g thi n loo p o fHenl e wher e diamete rbecome s narrowe r(se e sectio n IV-A) .

A 3

### Th e specificityofth e mal e rat respons e has bee n teste d t o a limite d

exten t i n a numbe ro

fothe rspecies , wit h n o evidence o fhyalin e drople t nephropath yi n dogs , guine a pigs , hamsters , o rmonkeys

. Sinc e thes e specie s (an d th e mous e an d femal e rat) hav e protein s simila ri n structur e t o ots-g

. ,h e ,ac k

0 ( 

### damag e t o thei rkidneycell s i s consisten t wit h th e presumptio n that th e specifi cO L- g produce d b yth e live rofmal e rat s i s necessar yfo rth e expressio n o

fth e rena l effects

. Mal e rat s o

fth e NB R strai n provid e a uniqu e opportunityfo rtestin g th e ots- g hypothesi s sinc e thi s anima l ha s n o detectabl e level s ofhepati c messenge rRN Afo ra^-g

. Unde rcondition s o fexposure tha t produce d ots- g nephropath yi n mal e rat s o fothe rstrains , severa l chemical s adminis tere d t o th e NB R rat di d no t induc e detectabl e accumulatio n o fots- g i n th e rena l tubules

. Mice an d femal e rat s expose d t o Os-g-inducer s i n chroni cbioassay s di d no t develo p a n increase d incidenc e o frena l tubul e tumors

. I n contrast , mal e rat s develope d a dose-dependen t neoplasti crespons e i n th e kidney

. Additiona l experimentatio n usin g a nitrosamin e a s th e initiato ro fcance ran d a n cL^g-induce ra s th e promote rals o suppor t th e observatio n tha t OL- g i s involve d i n th e proces s leadin g t o rena l tubul e tumor s i n th e mal e rat

. frron e o fthes e studies , th e promotio n potentia l o fa n a^g-induce ri n NB R rat s wa s contraste d wit h th e respons e i n a conventiona l strain , th e F34 4 rat

. Consis ten t wit h th e hypothesi s tha t a_- g i s necessar yt o induc e a response , th e promote rdi d not enhanc e renaitubul e tumo rformatio n i n th e ots-g-de ficien t NB R rat , bu t i t di d promot e rena l tubul e tumo rformatio n i n th e F34 4 rat

. I t i s clea rthat no t al l rena l tubul e cance ri n laborator yanimal s occurs throug h th e hypothesize d cx^- g sequence . Othe rinducers ofroden t rena l tubul e cance rare wel l known

. Thes e include , fo rexample , certai n nitrosamine s i n th e ra

t an d mous e an d diethylstilbestro l i n hamsters

. I n general , thes e prototypi crena l carcinogen s are activ e i n bot h male s an d females

. Th e acut e nephrotoxi cchange s i n th e rena l tubule s includ e mil d lipi d drople t accumulatio n an d scattere d single-cel l necrosis , bu t hyalin e drople t accumulatio n an d it s specifi cassociate d nephropath yar e no t characteristic

. Base d o n availabl e information , a^-g-inducers appea rt o hav e addi tiona l feature s that distinguis h the m fro m othe rroden t kidne ycarcinogens , suc h a s th e nitrosamines

. Alphas-globuli n inducer s appea rt o b e nongenotoxic , o ronl ymarginall yso , suggestin g that th e mechanis m fo r tumo rinductio n doe s no t depen d o n direc t geneti cinjury

. S o far , th e incidenc e o

frena l tumors produce d i n th e mal e rat byots- g inducers ha s bee n relativel ylow , occurrin g lat e i n life , an d metastasizin g rarely

. I n contrast , certai n roden t carcinogen s induce a hig h incidenc e ofkidne y tumor s afte ra s littl e as a singl e dose

. Distributio n studie s o t compound s an d informatio n o n chemica l bindin g t o o^ g indicat e that , o

fth e tota l chemica l administere d t o th e animal , onl y a smal l portio n ot th e metabolite s (possibl yth e paren t compound ) ca n accoun t fo ral l o

fth e rx, u 

### - g accumulation

. Considerabl e amount s ofth e

C M 

### chemica l an d othe rmetabolite s ma yals o b e presen t i n th e mal e ra t kid n no t boun d t o ots-g

. Thes e 0,he r

### moietie s may . at times , caus e toxi ceffe i n th e kidney , possibl yeve n cancer , that ar e unrelate d t o th e accumula t o fOs-g

. Suc h informatio n doe s not preclud e a determinatio n tha t ots- g sequence i s involve d i n som e manne rwit h th e rena l tumo rrespo n XVI . Scienc e Policy Statemen t Base d o n th e analysi s o

fth e scientifi cliteratur e i n Part s I throug h III , RA F Technica l Pane l reache d thre e majo rconclusions

. First , th e seque l o fevent s propose d t o lin k OL- g accumulatio n t o nephropath yan d r e tubul e tumors i n th e mal e ra t i s plausible , althoug h not totall yproven . Second , th e ots- g respons e followin g chemica l administratio n app e t o b e uniqu e t o th e mal e rat

. Eve n thoug h closel yrelate d protein s presen t i n othe rspecies , there i s n o evidenc e tha t thes e specie s respo n ots- g inducer

s i n a manne rsimila rt o th e mal e rat

. Third , th e mal e rat kidne yrespons e t o chemical s tha t induce a accumulatio n i s probabl ynot relevan t t o human s fo rpurpose s o f assessment

. Th e RA F Technica l Panel' s finding s provid e th e basi s fo ra tw o par t E scienc e polic

ystatemen t regardin g us e o fdat a o n mal e rat rena l tu t tumors fo rhuma n risk

assessment

. Thi s scienc e polic yapplie s t o indfvk chemical s o rchemica l mixtures

. (1 ) Mal e rat rena l tubul e tumor s arisin g a s a resul t o fa proces s Involvin g ots- g accumulatio n d o no t contribut e t o th e qualitative welght-of-evldenc e tha t a chemica l pose s a huma n carcinogeni c hazard . Suc h tumor s are not Include d I n dose-respons e extrapolation s fo rth e estimatio n o f huma n carcinogeni c risk . (2 ) I fa chemica l Induce s o^ g accumulatio n i n mal e rats , th e associate d nephropath y I s no t use d a s a n endpoin t fo r determinin g non-carcinogeni c hazard . Estimate s o fnon carclnogenl c ris k

ar e base d o n othe rendpoints . Eve n whe n chemicall yinduce d OL-g-relate d kidne ytumor s present , othe rtumors i n th e mal e ra t an d an ytumo ri n othe rexp o laborator yanimal s ma

yb e importan t i n evaluatin g th e carcinogeni cpo tia l ofth e chemical

. Likewise , th e rol e ofchemicall yinduce d ot^- g accu i latio n i n th e inductio n o

frena l tubul e tumor s i n th e mal e rat i s asse s independentl yo

fevaluation s mad e regardin g tumors a t othe rsites

. XVII . Guidanc e fo rEvaluatin g Chemicall y Induce d Mal e Ra t Re m Tubul e Tumor s To determin e th e appropriat e us e o fth e dat a fo rEP

Aris k assessm e chemical s inducin g rena l tub u l e t u mor s i n th e mal e ra

t ar e examine d i n te i o fthre e categories

. (1 ) Th e Os- g sequence o fevent s account s fo rth e rena l tumors

. 8 5(2 ) Othe rpotentia l carcinogeni c processe s accoun t fo rth e rena l

tumors . (3 ) Th e OL u -g-associated event s occu ri n th e presenc e o fothe r potentia l carcinogeni c processes , bot h o fwhic h resul t i n rena l tumors . Tw o question s nee d to b e answered . Th e first an d simplest questio n i s whethe ro rnot th e 0L.o

- g proces s i s involve d i n th e tumo rdevelopment . Th e secon d mor e difficul t question , give n a n affirmative answe rt o th e first , i s th e exten t t o whic h a^-g-associate d events , rathe rtha n othe rprocesses , accoun t fo rth e tumo rincrease . A determinatio n o

fth e exten t t o whic h th e o^ g proces s i s involve d i n tumo rdevelopmen t require s a substantia l database , an d no t just a limite d se t o finformatio n confine d to th e mal e rat . Fo rexample , cance rbioassay dat a ar e neede d fro m th e mous e an d th e femal e rat to b e abl e to demon strate that th e rena l tumors are male-rat specific . Eve n t o answe rth e firs t questio n affirmatively , informatio n fro m toxicit ystudie s must demonstrate whethe ro rno

t th e o^ g proces s i s operative (se e sectio n XVII- A below) . I n th e absenc e o

fthi s minimu m information , ther e i s n o basi s fo rjudgin g th e applicabilit

yo fth e OL,

u - g process , an d i t woul d b e assume d that th e mal e rat rena l tumors are relevan t fo rris k assessmen t purposes . Additiona l dat a ar e necessar y(se e sectio n XVI I B below) t o answe rth e secon d questio n an d t o assig n a chemica l t o categorie s 1 , 2 . o r3 . A. Renal

Tubule Tumors

In Male Rats and Alphas-globulin Accumulation Th e followin g informatio n fro m adequatel yconducte d studie s o fmal e rat s shows that th e OL. u - g proces s coul d b e a facto ri n th e observe d rena l effects ; a n affirmative respons e i n eac h o fth e thre e categorie s i s required . Ifdat a d o no

t mee t th e criteri a i n an y on e category , th e availabl e rena l tumo r dat a shoul d b e analyze d i n accordanc e wit h standar d ris k assessmen t principles . (1 ) Increase d numbe ran d size o fhyalin e droplet s i n rena l proxima l tubul e cell s o

ftreate d mal e rats Th e abnorma l accumulatio n o fhyalin e droplet s i n th e P 2 segmen t o fth e rena l tubul e i s necessaryt o attribute th e rena l tubul e tumor s t o th e ots- g sequenc e o

fevents . Thi s findin g help s differentiat e o^ g inducers fro m chemical s that produc e rena l tubul e tumors throug h othe rmeans . (2 ) Accumulatin g protei n i n th e hyalin e droplet s i s 0L, u - g Hyalin e droplet accumulatio n i s a nonspecifi c respons e t o protei n overloa d i n th e rena l tubul e an d ma yno t b e du e t o c^ g (e.g. , a s wit h chlorothalonil) . Therefore , i t i s necessar

yt o demonstrat e that ots- g ac " counts fo rth e hyalin e drople t accumulatio n foun d i n th e mal e rat . (3 ) Additiona l aspect s o fth e pathologica l sequenc e o flesion s associate d wit h a 2o - g nephropath yar e present . 86 Typica l lesion s include : single-cel l necrosis , exfoliatio n o fepithelia l cell s into th e proxima l tubula rlumen , formatio n ofgranula rcasts , linea rmineral izatio n o fpapillar ytubules , an d tubul e hyperplasia . Ifth e respons e i s mild , al l o fthes e lesion s maynot b e observed ; however , som e element s consis ten t wit h th e pathologica l sequenc e must b e demonstrate d t o b e present . B. Additional

Information

Useful for the Analysis I fth e precedin g analysi s (sectio n XVII-A ) indicate s tha t th e OL- g proces s i s operative , the n othe rinformatio n i s reviewe d t o determin e iftn e rena l effect s ar e solel ya^ g associated , a combinatio n o fth e O L - g proces s an d othe rpotentia l carcinogeni c processes , o rdu e primaril yt o othe r processes . Man ykind s o finformatio n ca n assist i n confirmin g tha t chemi call yinduce d ots- g accumulatio n i s involve d i n th e rena l tumo rrespons e o r that othe rprocesse s cannot b e rule d out . Som e o fthes e finding s ar e liste d below ; th e informatio n ma ynot alway s b e available , no rshoul d thi s list b e considere d exhaustive . Hypothesis-testin g data : Dat a fro m specialize d test s ca n greatl y increas e confidenc e tha t th e a^- g sequenc e i s involve d i n th e rena l tubul e tumo rresponse . Suc h informatio n migh t include : modificatio n ofth e nephrotoxi c respons e throug h us e o fth e NB R rat , o rmanipulatio n o fse x hormone s (e.g. , androgens ) o ra^ g level s (e.g. , o^- g administratio n t o femal e rats) . Othe rinformatio n migh t includ e initiation-promotio n studie s comparin g male s o

fth e NB R strai n wit h male s o fothe rra

t strains . Additiona l biochemica l information : Certai n i n vivo an d i n vitr o dat a hel p characteriz e a chemica l a s on e that induce s accumulatio n ofOL^-g . Suc h informatio n migh t include : reversibl e bindin g o fth e chemica l (o rmetabo lites ) t o Os-g , reductio n i n th e lysosoma l degradatio n o fth e OL-g-complex , an d dispositio n studie s demonstratin g sex - an d species-specifi c retentio n o f th e tes t compoun d i n th e mal e ra t kidney . Sustaine d cel l divisio n i n th e proxima l tubul e o fth e mal e rat : A sustaine d increas e i n cel l replicatio n i n th e P 2 segmen t o fth e rena l tubul e at dose s use d i n th e cance rbioassayan d a dose-relate d increas e in atypica l hyperplasi a ofth e rena l tubul e i s consisten t wit h th e a - g process , espe ciall yifothe rlaborator yanimal s wer e teste d an d di d no t sho wsimila r responses . Thes e endpoints ar e nonspecifi c fo rOL- g inducers , however , sinc e othe rrena l carcinogen s ma yals o affect th e P 2 segmen t ofth e rena l tubule . Structure-activit

yrelationships : Structure-activit yrelationship s fo rchemi cal s that induc e a.- g accumulatio n i n th e mal e rat kidneyare no t wel l defined , althoug h ther e appea rt o b e dimensiona l requirement s t o fi t th e protei n pocket , a requiremen t fo ra degre e o flipophilicity . an d a nee d fo ra n electronegative ato m i n th e molecul e o rits active metabolite . Othe r structura l feature s migh t suggest that a chemica l belong s t o a differen t clas s o fsuspecte d carcinogens . Covalen t bindin g to macromolecules : Som e inducers o frena l tubul e cance ri n rodents (eg , nitrosamines ) ar e know n t o bin d covalentl yto DN A 8 7

### o rothe rmacromolecules

. Others d o no t appea rt o bin d t o DN A(e.g. , isophorone ) suggestin g tha t suc h informatio n ma yassist i n distinguishin g differen t processe s leadin g t o rena l cancer . Genotoxicity : Althoug h rena l tubul e neoplasi a associate d wit h clearl y genotoxi cchemical s i s a wel l know n response , informatio n t o dat e support s a conclusio n that OL^-

g inducers are essentiall ynongenotoxi can d d o no t depen d o n direct geneti cinjur yfo rth e productio n o

ftumors

. Thus , informatio n o n potentia l genotoxicityi n a standard batter yofshort-ter m test s relevan t t o th e evaluatio n o fpotentia l carcinogenicit yprovide s a possibl e device fo rhelpin g t o distinguis h betwee n thes e processes . Nephrotoxicity : Chroni cprogressiv e nephropath y(CPN ) i n th e agin g mal e rat ca n complicat e th e analysi s o fothe rrena l lesions

. However , nephrotoxicit

yi n th e mal e rat not attributabl e t o eithe rCP N o rots- g accumulation , o ra nephrotoxi crespons e i n th e femal e rat o rth e mouse , suggests that th e possibilityofothe rprocesse s leadin g t o rena l cance r shoul d b e considered

. Anima l bioassaydat a i n othe rspecies- , sex-combinations : Th e ot^-g syndrom e i s specifi ct o th e mal e rat

. Positive cance rresponse s i n th e rena l tubul e i n femal e rats , mic e o feithe rsex , o ran yothe rlaborator yanima l impl y tha t th e OL- g syndrom e aton e doe s not accoun t fo rth e rena l tubul e tumo r respons e i n th e mal e rats

. Confidenc e i n determinin g whic h ofth e thre e categorie s applie s de pend s o n th e comprehensivenes s an d consistenc yofavailabl e data

. I fal t th e dat a (two species , tw o sexcombinatio n bioassay , al l element s i n XVII A

. an d additiona l informatio n suc h a s tha t describe d i n XVII-B ) are consis ten t wit h a rol e fo rchemicall yinduce d ots-g

. there i s a hig h degree o f confidence that th e OL- g syndrome , alone , accounts fo rth e rena l tubul e tumors

. I n contrast , ifInformatio n fro m adequat e testin g I s Inconsisten t wit h th e ots- g syndrom e (e.g. , rena l tubul e tumor s are presen t i n femal e rat s o r mice) , othe rcarcinogeni cprocesse s probabl yaccoun t fo ral l o rmos t o fth e rena l tumors

. Sometimes , th e informatio n wil l indicat e tha t more tha n on e carcinogeni cproces s i s occurring ; i n thes e cases , a s a minimum , th a criteri a i n suppor t ofOL- g involvemen t (sectio n XVII-A) are present , bu t ther e i s als o evidenc e consisten t wit h othe rmechanisms

. Decision s o n th e applicability ofth e thre e categorie s ca n onl yb e mad e o n a case-by-cas e basis , takin g al l o fth e informatio n int o account

. Whateve rth e finding , th e risk assesso r shoul d clearl ydelineat e an d thoroughl ydocumen t th e basi s fo ran ydeci sion s made

. C. Use of the Data for Risk Assessment Once a decisio n o n th e applicabilityo fth e thre e categorie s i s made , I t become s possibl e t o determin e ho wth e respons e I n th e mal e ra t rena l tubul e woul d appl yt o evaluatin g huma n hazard an d t o estimatin g huma n cance rrisk. I n general , th e followin g guidance applies , recognizin g tha t tumors occurrin g a

t othe rsite s i n laborator yanimal s administere d com pound s that induce a^- g accumulatio n i n th e mal e rat wil l b e judge d o n thei r ow n merits

. 8 8 

### Compound s producin g rena l tubul e tumor s I n mal e rat s attribut abl e solel yt o chemicall yInduce d a Ju 

### - g accumulation : thes e rena l tubul e tumors wil l no t b e use d fo rhuma n cance rhazar d identificatio n o rfo rdose respons e extrapolations

. Compound s producin g rena l tubul e tumor s that ar e not linke d t o ots- g accumulation : thes e rena l tubul e tumors are a n appropriat e endpoin t fo rhuma n hazar d identificatio n an d ar e considered , alon g wit h othe r appropriat e endpoints , fo rquantitativ e risk estimation

. Compound s producin g som e rena l tubul a tumor s I n mal e rats attributabl e t o th e OL- g proces s an d som e attributabl e t o othe rcarci nogeni c procosses.'T n general , th e informatio n neede d t o make a quanti tativ e determinatio n o fth e relative contributio n o feac h proces s t o tumo r developmen t wil l no t b e available

. Thus , eve n thoug h th e informatio n o n th e rena l tubul e tumor s remain s relevan t fo rpurpose s o fhazar d identification , a meaningfu l dose-respons e estimat e base d o n rena l tubul e tumors I n th e mal e ra t i s generall yno t possibl e an d shoul d not b e performed

. I fther e i s enoug h informatio n t o determin e th e relativ e contributio n o feac h proces s t o th e overal l rena l tubul e cance rrespons e i n mal e rats , th e non-ots-g-induce d componen t ma

yb e used , a s appropriate , fo rdose-respons e evaluatio n as wel l a s hazar d identification

. XVIII . Nephropath ya s a Toxi cEndpoin t I fa compoun d induce s a.- g accumulatio n i n hyalin e droplets , th e associate d nephropath yi n mal e rat s i s not a n appropriat e endpoin t t o determin e noncance r(systemic) effect s potentiall yoccurrin g i n humans

. Lfcewise , quantitativ e estimate s o fnoncance rris k

(e.g. , referenc e dose s an d margin-of-exposure determinations ) ar e base d o n othe rendpoints

. I t shoul d not b e anticipate d that a compoun d tha t produce s nephropath y i n th e mal e rat throug h th e sequence ofevent s beginnin g wit h th e accumu latio n o fa-- g wil l always b e foun d t o induc e rena l tubul e tumors i n th e mal e rat

. Th e abilityt o detect rena l tumors depend s o n man yfeature s that ma y not b e presen t i n an yindividua l experiment , e.g. , sufficien t dos e t o Induce effec t withou t eart

ydeath s ofth e animals , competin g toxicityfro m othe r moietie s not boun d t o a^-g

. insufficien t lengt h o fexposure orfollowup , an d incomplet e histopathology

. Eve n i n th e absenc e o frena l tubul e tumors i n th e mal e rat , i fth e sequence o flesion s characteristi cofth e a^- g syndrom e ar e present , th e associate d nephropath yi n th e mal e rat doe s no t contribut e t o determination s o fnoncarcinogeni chazard o rrisk.

8 9P.5 3 

# 4

Appmndbt

•Hon Neopftk

Effects

of Hyaline Droplet Mucin Table A-1. Subatanem

that have Induced

Hyaline Droplet Accumuiabon

and/or Elevated

Lavrnk of Alpha,, -Globulin

In Ranal Proximal Tubulee of Rata

Substanca/CtMmtca l Evidenc e forexeoerMtto n ofhyaln a dropfo U I n rana l proxima l tubul e cat o Evidenc e fo rIncrease d rana l alpha^-oJobuM n level s

Unleade d gasolin e

2,2,4-Trimethyi pentan e JP- 4 jet fue l (mixe d distillat e hydrocarbons)

JP- 5 je t fue l (mixe d distillat e hydrocarbons

)

Diese l fuel , marin e JP-1 0 syntheti c je t fue l + (exohexahydro-4,7- methanoindan ) N R Haide retal . (1964 ) Thoma s etaj . (1985 ) Murt y etaJ . (1988 ) Gar g etal . (1988 ) Stonard etal . (1985,1986 ) Brune r(1984 ) MacNaughto n an d Uddi n (1984 ) Parke retal . (1961 ) Brune r(1964 ) Gaworskietal . (1984 ) MacNaughto n an d Uddi n (1984 ) Brune r(1984 ) Gaworski etal . (1985a ) MacNaughto n an d Udtf n (1984 ) Matt e etal . (1988 ) •

•

N R

N R

N R N R N R

-

N R

N R

N R N R Olso n etal . (1987 )

Gar g etal . (1988 )

Stonar d etal . (1986 ) Charbonnea u etal . (1967 ) Loc k etal . (1987b )

Table A-1. (oonl)

Substance/Chemica l Evidenc e fo rexacerbatio n ofhyalin e droplet s I n rena l proxima l tubul e cett a Male s Female s Reference s Evidenc e fo rincrease d rena l alpha2 U -oJobull n level s Male s Female s RJ-5 syntheti c jet fue l (hydrogenated dimer

s o fnorbomadwne

) JP- 7 distillat

e jet fue l

JP-T S distillat

e je t fue l

Stoddar d solvent

c 10" c 1 1 andC 1 0 -C 1 2 isopararfini c solvent

s (saturate d aliphati c hydrocarbons

) Decali n

Tetrai n MacNaughto n an d Uddi n (1984 ) N R

N R Brune r(1990 , unpublished N R

data ) Alde n (1989 ) Brune r(1990 , unpublished N R data ) Alde n (1989 ) Phillip s an d Cockrel l N R (1984 ) Phillip s an d Cockrel l + (1964 ) Via u etal . (1986 )

Alde n etal . (1984,1985 ) • Brune r(1984 ) Gaworski etal . (1985b ) Kanerv a etal . (1987a ) Ston e etal . (1987 ) Serve etal . (1988 ) N R N R

N R

N R

NR Reference s

Via u etal . (1986 )

Alde n etal . (1964 , 1985 ) Kanerv a etal . (1987b )

NRP.54

_

Table A-1. (cont)

Substance/Chemica l Evidenc e forexacerbatio n o fhyain e droplet s b i rana l proxima l tubul e cell s Male a Female s Reference s Evidenc e fo rIncrease d rana l alpha 2u i)lobuH n levei a Male a Female s Reference s

CO r o d-Limonen e

Pentachlorobenzen e 1,2,4,5-Tetrachtoro benzen e 1,4-DichJorobenzen e Tetrachloroethylen e (Perchtoroethylene

) Pentachloroethan e Hexachloroelhan e Isophoron e Lindan e N R Ridde retal . (1987 ) Kanerva etal . (1987a ) NT P (1990 ) Lehman-McKeema n et al . (1989 ) Web b etal . (1989 ) NT P (1991a ) NT P (1991b )

NT P (1987a ) Bomhar d etal . (1988 ) Charbonnea u e t al . (1989 ) Goldsworthyetal . (1988a ) Gree n etaJ . (1990 ) Goldsworthye t al . (1988a ) NT P (1989 ) Strasse retal . (1988 ) Dietoc h an d Swenber g (1990,1991b ) •

NR NR

•

•

• N R +

•• • NR NR NR

-

- N R N R • Ridde retal . (1968 )

Lehman-McKeema n e t a l (1969 ) Web b etal . (1989 )

Charbonnea u e t al . (198 9

Goldsworthye

t al . (1968 a Goldsworthyetal . (1988a )

Strasse retal . (1988 ) Dietric h an d Swenberg (1990,1991b )

Table A-1. (eont)

Substance/Chemica l Evidenc e forexacerbatio n o fhyain e droplet s I n rena l proxima l tubul e eeOa Pemala a Reference e cvtdonc o forinoreaae d rena l alpha 2l| .globut n levei a Male a Female s

t o Dimethy l mettryt

phosphonat

e Methy l isobuty l keton e Methy l isoamy l keton e Diisobuty l keton e BW540 C (3-methylamino-1 - (3-dTflucfomethyl phenyl)-2-pyra2oline) BW58 C (mixtur e o fisomeri c ci s an d tran s form s o f2-(4M-butytcyctohexyl ) -3-hydroxy- 1 -4-naphtho quinone ) Levamisol e (levoisome ro

f2,3,5,6 - tetrahydro-6-phenyl- *nidazo-(2,l-b)thiazole ) NT P (1987b ) PhUp s ate . (1987 ) Katz etal . (1986 ) Dod d eta) . (1987 )

Rea d etal . (1988 )

Rea d etal . (1988 ) N R N R NR N R

+

• N R N R N R N R N R

NR Rea d eta) . (1988 )

Rea d eta) . (1968 )

Rea d etaJ . (1988 ) N R Rea d etal . (1968 )P.5 5

Table A-1. (cont)

Substance/Chemica l Evidenc e fo rexacerbatio n o fhyalin e droplet s I n rena l proxima l tubul e cell s Evidenc e fo rIncrease d rena l alphs 2u -globull n levei a Mal a Female s Reference s Male s Female s Reference s Gabapenti n Dominic * e t al . (1990 ) Domintcketal . (1990 ) 3,5,5-Trimethyl-hexanoi c ad d derivatives Tridecy l acetate Isopropylcydohexan e 1,3,6-Thcyanohexan e +

+ + + NR

N R N R

- Lehman-McKeema n et al .

(1991 ) Daughert yetal . (1990 ) Hermingse n e t al . (1988 ) Bamet t etal . (1987 ) Johnso n (1987 ) +

+ + N R N R

N R N R N R Lehman-McKeema n e t a l

(1991 ) Daughert y etal . (1990 ) Hermingse n e t aJ . (1988 )

•Positiv

e Negativ e N R « No t reported

Appendix

- Non-Neoplastic

Effect s of

Hyaline Droplet Inducer* Table A-1 Non-Neoplaatk: Finding*

Reported

2-Year Studlaa on Ten Selected

Substance s that

Produce

Renal Tubule Tumor* In Rata Chemleal/Substane e

t o 1,4-Dichlorobenzen e species . F34 4 rat

s B6C3F1mic e route : gavag e ret : NTP-TR-31 9 (NTP , 1987a) Dimethy l methyiphosphonat e species : F344rat s B6C3F1mic e route : gavag e ret : NTP-TR-32 3 (NTP , 1987b)

Hexachloroethan e species . Osborn e Mende l ra t B6C3Flmic e route : gavag e ref : NTP-TR-6 8 (NCI , 1978b ) Non-Neoplasti c lealon s Rena l damag e wa s no

t observe d i n femal e rats o rmice i n 1 3 woo k studtes . Ce l degeneratio n o rnecrosi s o

ftubul e epitheliu m was observe d i n mal e rats i n a 13-wee k study . I n 2-yea r studies , th e severit

yo fnephropath ywa s greate ri n mal e rats tha n iemal e rats . Nephropath y characterize d b

ydegeneratio n an d regeneratio n o frena l tubul e epithelium , tubul e dilatio n wit h attenuatio n an d atrophyo fth e epithelium , granula rcasts i n tubules o fth e oute rstripe o fth e medusa , thickenin g o fbasemen t membranes , an d minima J accumulatio n o

finterstitia l collagen . Rena l tubula rregenerabo n wa s note d i n femal e mice . Neohropatfi y increase d wit h dos e i n mal e mice an d Iemal e rats . Kidney s o fdose d mat

e rats but no

t o fdose d mal e mice ha d varyin g degree s o ftubul e cel l re generation , hyalin e drople t degeneration , an d cellula rinfiltrate (1 3 wks) . A t 2 years , th e averag e sevent

yo fnephropathyincrease d an d calcificatio n wa s observe d i n th e collectin g tubules o

fth e rena l pelvi s o

fmal e rats . Th e nephropath ywa s characterize d b y degeneratio n o

fth e tubul e epithelium , tubul e dilatio n wit h attenuatio n an d atrop yo t th e epithelium , granula rcasts i n th e tubules o fth e oute rstripe o

fth e oute rmedulla , thickenin g o

fbasemen t membranes , minima l to mil d accumulatio n o finterstitia l cotagen , an d minima l to mil d inflammator yce l infiltrates . Th e increas e i n severit yo f nephropath ywa s ranked , contro l to hig h dose . 1.9 , 2.5 , an d 2.8 , o n scal e o f1 to 4 . Toxi c tubul e nephropath ywa s observe d i n al l groups o ftreate d rat

s an d i n mal a an d femal e mice . Th e nephropath yi n rats wa s characterize d b ydegeneration , necrosis , an d th e presenc e o flarg e hyperchromatt

c regeneratin g epithelia l eels . Overlyin g th e tubul e lesion s wer e chroni c interstitia l fibrosi s an d nephritis , foca l pyelonephritis , tubula rectasia , cast formation , an d foca l glomerulosclerosis . I n mice , nephropath ywas characterize d b y degeneratio n o

t affecte d tubules containin g hyalin e casts . Th e kidne yofte n showed infiltratio n o

t inflammator

ycells , fibrosis , an d calciu m deposition . Th e incidences o f toxic nephropath ywere highe ri n mic e tha n i n rats .P.5 6

Table A-2. (cont) Chemical/Substanc e Non-Neopiastl e lesion s

< o en Hexachloroethan e (cont ) species : F34 4 rats route : gavag e ref : NTP-TR-36 1 (NTP , 1989 )

Isophoron e species : F344ra t B6C3F 1 mouse route : gavag e ref : NTP-TR-29 1 (NTP . 1896a ) d-Limonen e species : F344rat s B6C3F1mio e route : gavag e ref : NTP-TR-34 7 (NTP . 1990 ) Pentachloroethan e species : F344rat s B6C3Flmic e route : gavag e ref. : NTP-TR-23 2 (NTP . 1983 ) Nephropath yobserve d i n nearl yal l males ; overal l averag e severit ymi d i n vehicl e control s an d mi d t o moderate i n dose d males . Incidence an d severit yo fnephropath ywa s increase d i n dose d female s relative to controls . Nephropath yi n eac h se xconsisted o

ftubul e ce l degeneration , regeneratio n an d dilation , atrophy , glomerulosclerosis , interstitia l fibrosis , an d chroni c inflammation . Linea rmineralizatio n o fth e rena l papita e showe d a doseretate d increas e i n mal e rats . Hyperplasi a o fpelvi c transitiona l epitheliu m wa s increased . Incidences o

t mineralizatio n an d pelvi c epithelia l hyperplasi a were no t increase d i n females . Tubula rceH mineralizatio n was increase d i n dose d mal e rats but no t i n dose d femal e rats . Thi s lesio n wa s characterize d b ybasophili c aggregate s ot mineral s mos t ofte n foun d i n th e medullar ycolectin g ducts an d occurred coincidentalt y wit h lesion s o

fchroni c nephropathy . Th e incidence o i nephropath ywa s simila ri n dose d an d vehicl e contro l mal e rats , th e severit y wa s greate ri n lo

wdos e mates . Hyperplasi a o fth e rena l pelvi s wa s observe d i n dose d mal e rats bu t no

t i n vehicl e controls . Increase d severit

yo fnephropathyi n mal e rats a t 1 3 week s characterize d b

ydegeneratio n o fepithelia l eel s i n convoluted tubules , granula rcasts i n th e oute rmedulla , an d epitneia t regeneration . N o lesion s i n femal e rats . Increas e i n severit yo fnephropath y an d inea rdeposit

s o fmineral s i n th e rena l medusa an d papll a i n mal e rats i n 2-yea rstudies . Chronic , diffuse inflammation , distinguishabl e fro m nephropath ysee n i n agin g F34 4 rats , wer e found i n mal e rats i n a significant dose-relate d increase . Interstitia l fibrosi s an d tubul e elatio n mor

e severe tha n i n old-ag e nephropathy . Mineralizatio n o fth e rena l papi a observe d a t increase d incidences i n dose d mal e rats . Som e dsate d tubules wit h gian t eel s an d casts wer e observed . N o inckatio n o frena l toxicity wa s reported fo r femal e rats o rmice .

Table A-2 (cont) Chemical/Sub s lance Nonneoplasti c lesion s Tetrachloroethylen e species : F34 4 rat

s B6C3F 1 mic

e route : inhalatio n ref : NTP-TR-31 1 (NTP , 1966b ) Unleade d gasolin e species . F344ra t route : inhalatio n •ef : USEPA , 198 7

( O

Chlorothaloni l species : Osborne-Mende l rats BSCSFImie e route : d» t ref : NTP-TR-4 1 (NCI . 1978a ) Trichloroethylen e species : AC I rat

s Augus t rat s Marsha l rat

s Osborne-Mende l rat

s route: gavag e ref : NTP-TR-27 3 Bot h mal e an d femal e rats exhibite d rena l tubul e cel l karyomegaly . Karyomegal yals o occurred i n mice . Cas t formatio n wa s noted i n mal e an d femal e mice . Tubul e cel l hyperplasi a wa s see n i n mal e rat

s an d mal e mice .

Dose-relate d kidne

ylesion s a

t & 6 month s i n males included foca l tubul e basophili a an d tubula reasts a t th e cortcomedullar y junction . A n interrelate d increas e i n th e incidence o frena l pelvi s mineralizatio n was als o reported a t 1 2 months , 1 8 months , an d termina l sacrifice . Progressiv

e glomerulonephrosi s wa s reporte d i n on e high-dose mal e a t 1 2 months ; th e incidenc

e was highe rat 1 8 month s but wa s dose-related ; at th e fina l sacrifice , nearl y a l mal e rats exhibited thi s lesio n (MacFarian d e t al. , 1984) . Karyomegaly , i.e. , ver ylarg e nucle i withi n cell s o

ftubul e epithelium , firs

t note d a t 1 2 months ; a t 1 8 months , mor e numerou s karyomegali c cell s were observe d i n treate d males , particularl yi n th e high-dose grou p (UAREP . 1983) . N o non-neoplasti c rena l lesion s wer e reported i n th e NT P bioassays .

Cytomegaf

y noted i n males an d female s o fal l strains . Toxi c nephropath yincrease d i n bot h sexes o fal l strains . Calcificatio n was produced i n kidney s o fAC I mal e an d femal e rats .

## Reference s

AkerstrOm , B . an d Logdberg , L . (1990 ) A n intriguin g membero fth e lipocali n protei n famity : OL-microglobulin . Trend s Biochem . Sci . 15:240-243 . Alden , C.L . (1986 ) A reviewo funiqu e mal e rat hydrocarbo n nephropathy . Toxicologi c Pathol . 14:109-111 . Alden , C.L . (1989 ) Mal e rat specifi c alpha , globuli n nephropath yan d rena l tumorigenesis . In : Bach , PH. , an d LOCK , EA . (eds) , Nephrotoxicity . I n Vitro t o I n Vivo . Animal s t o Man . Plenu m Press , Ne wYork . pp . 535-541 . Alden , C.L. , an d Frith , C.H . (1991 ) Toxicologi c patholog yo fth e kidney . In : Haschek , W.M. , an d Rousseaux , C.G . (eds) , Handboo k o fToxicologi c Pathology . Academi c Press , Ne wYork . I n press . Alden , C.L. , Kanerva , R.L. , Ridder , G. , an d Stone . L.C . (1984 ) Th e pathogenesi s o

fth e nephrotoxicit

yo fvolatil e hydrocarbon s i n th e mal e rat . In : Mehlman.M.A.Hemsireet.G.P. , Thorpe . J.J. , an d Weaver , N.K . (eds.) . Advance s i n Mode m Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton , Ne wJersey , pp . 107-120 . Alden , C.L. , Ridder , G. . Stone , L. . an d Kanerva , R.L . (1985 ) Patholog yo f petrochemica l fuel s i n mal e rats . Acut e toxicity . In : Bach , P.H. , an d Lock , E.A . (eds.) , Rena l Heterogeneityan d Targe t Cel l Toxicity . Joh n Wileyan d Sons , Ne wYork , pp . 461-472 . Arnold , F.J. , an d Meyerson , L.R . (1990 ) Radia l immunodiffusio n assa yfo r rat oL-aci d glycoprotein . Pharmacol . Biochem . Behavio r37:485-491 . Asal , N.R. , Risser , D.R. , Kadamani , S. , Geyer , R. , Lee , E.T. , an d Chemg , N . (1988a ) Ris k

factors i n rena l cel l carcinoma : I . Methodology , demographics , tobacco , beverag e use , an d obesity . Cance rDetectio n Preventio n 11:359-377 . Asal , N.R. , Geyer , J.R. , Risser , DR. , Lee , E.T. , Kadamani , S. , an d Chemg , N . (1988b ) Ris k

factor s i n rena l cel l carcinom a II . Medica l history , occupation , multivariate analysis , an d conclusions . Cance rDetectio n an d Preventio n 13:263-279 . Ashby , J. , an d Tennant , R.W . (1988 ) Chemica l structure . Salmonell a mutagenicityan d exten t o fcarcinogenicitya s indicators o fgenotoxi c carcinogenesi s amon g 22 2 chemical s teste d i n rodent s by th e U.S . NCI / NTP . Mutat . Res . 204:17-115 . Askergren , A . (1986 ) Solvent s an d th e kidney . Progr . Clin . Biol . Res . 220 : 155-167 . Bachmann , S. . Sakai , T. , an d Kriz , W. (1986 ) Nephro n an d collectin g duc t structure i n th e kidney , rat . In : Jones , T.C. , Mohr , U. . an d Hunt , RD . (eds) . Urinar

ySystem . ILS I Monograph s o n Patholog yo fLaborator

y Animals . Springer-Verlag . NewYork , pp . 3-24 .

9 9Bannayam . G.A. . an d Lamm . D.L . (1980 ) Rena l cel l tumors . Pathol . Ann . 15 . par t 2 . 271-308 . Bamett . J.W. . Johnson , D.E. , Boothe . A.D. , an d Johannsen , F.R . (1987 ) Protei n accumulatio n a s th e initia l even t i n tricyanohexan e induce d nephropath y i n mal e rats . Toxicologist 7:2 7 (abstract) . Barrientos , A. , Ortuno . M.T. , Morales . J.M. , Martine z Tello , F. , an d Rodicio , J.L . (1977 ) Acute rena l failure afte rus e o fdiese l fue l a s shampoo . Arch . Intern . Med . 137 : 1217 . BarthokJ , S.W . (1979 ) Chroni c progressive nephropath y i n agin g rats . Toxicol . Pathol . 7: 1 -6 . Bennington , J.L. , Ferguson , BR . an d Campbell . P.B . (1968 ) Epidemiologi c studie s o

fcarcinom a o

fth e kidney . II . Associatio n o frena l adenom a wit h smoking . Cance r22 : 821-823 . Bennington , J.L. , an d Beckwith , J.B . (1975 ) Tumors o fth e Kidney . Rena l Pelvis , an d Ureter . Atla s o t Tumo rPathology . Secon d Series . Fascicl e 12 . Arme d Force s Institute o fPathology , Washington , D.C , pp . 93-199 . Bell , ET . (1950 ) Rena l Diseases . Le a an d Febiger , Philadelphia , p . 428 . Berggard , I . (1970 ) Plasm a protein s i n norma l huma n urine . In : Manuel . Y. , Revillard , J P. , an d Betuel . H . (eds.) . Protein s i n norma l an d pathologica l urine . S . Karger , Basel , pp . 7-19 . Bernard , A.M. , Lauwerys , R.R. . Noel , A. , Vandeleene . B. . an d Lambert . A . (1989 ) Urin e protei n 1 : a sexdependen t marke ro ftubula ro rglomerula r dysfunction . Clin . Chem . 35 : 2141-2142 . Blair , A , Decoufle , P. , an d Grauman , D . (1979 ) Cause s o fdeat h amon g laundr y an d dr y cleanin g workers . Am . J . Publi c Healt h 69:508-511 . Blair . A. , Stewart . PA , Tolbert . P.E. . Grauman , D. . Moran . F.X. , V aught , J. , an d Rayner , J . (1990 ) Cance ran d othe rcause s o fdeat h amon g a cohor t o fdr y cleaners . Brit . J . Industr . Med . 47:162-168 . Bomhard , E. . Luckhaus , G. . Voight . W-H. . an d Loeser , E . (1988 ) Inductio n o fligh t hydrocarbo n nephropath y by p-dichlorobenzene . Arch . Toxicol . 61 : 433-439 . Bomhard , E. . Marsmann , M. , Ruh l Fehlert , Ch. , an d Zywietz , A . (1990 ) Relationship s betwee n structure an d inductio n o fhyalin e drople t accumulatio n i n th e rena l cortexo fmal e rat s b

y aliphati c an d alicycli c hydrocarbons . Arch . Toxicol . 64 : 530-538 . Borghoff , S.J. , Strasser , J. , Charbonneau , M. , an d Swenberg , J.A . (1988 ) Analysi s o

f2,4.4-trimethyl-2-pentano l (TMP-OH ) bindin g t o mal e rat kidne y OL-globuli n (oL)andotherproteins . Toxicologis t 8:13 5 (abstract) . Borghoff , S.J. . Upton , P.B. , an d Swenberg , J.A . (1989 ) Characteristic s o f 2.4,4-trimethyl-2-pentano l (TMPOH ) bindin g t o o^-globuli n an d othe r compound s that caus e protei n droplet nephropathy . Toxicologist 9:7 9 (abstract) . Borghoff . S.J. . Short , B.G. . an d Swenberg , J.A . (1990 ) Biochemica l mechanism s an d pathobiolog y o fOL^-globuli n nephropathy . Ann . Rev . Pharmacol . Toxicol . 30:349-367 . Borghoff , S.J. , Miller , A.B. , Bowen . J.P. , an d Swenberg , J.A . (1991 ) Characteristic s o

fchemica l bindin g t o ot^-globuli n i n vitro - evaluatin g structure-activit

y relationships . Toxicol . Appl . Pharmaco l 107 * 228 - 238 . Boring , C.C. , Squires . T.S. . an d Tong . T . (1991 ) Cance rStatistics . ( 19-36 . Brooks . D.E . (1987 ) Th e majo randrogen-regulate d secretor y pro t th e ra t epididymi s bea rsequenc e homology wit h members o ft globuli n superfamily . Biochem . Internal . 14:235-240 . Brown , DP , an d Kaplan , S.D . (1987 ) Retrospective cohor t mortali r o fdr y cleane rworker s usin g perchloroethylene . J . Occup . M i 535-541 . (1987) . Brownson , R.C . (1988 ) A case-contro l study o frena l cel l carci n relatio n t o occupation , smoking , an d alcoho l consumption . Environ . Health . 43 : 238-241 . Bruckner , J.V. , Davis , B.D. , an d Blancato . J.N . (1989 ) Metabolism , t an d carcinogenicity o

ftrichloroethylene . CR C Critica l Rev . Tox i 31-50 . Bruner . R.H . (1984 ) Pathologi c finding s i n laborator y animal s ex p hydrocarbo n fuel s o

fmilitar y interest . In : Mehlman , M.A. , He r G.P. . Thorpe , J.J. . an d Weaver . N.K . (eds) . Advance s i n I Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPe l Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princet o Jersey , pp . 133-140 . Bruner , R.H.(1986 ) Chroni c consequenc e o falphas-globuli n neph r review . Proceedings , Toxicolog y Forum . Aspen , Colorado , Jul y 161-166 . Burnett , V.L. . Short , B.G. . an d Swenberg , J.A . (1989 ) Localizato r globuli n withi n protei n droplet s o fmal e ra t kidney : immunohistocf i usin g perfusion-fixed , GMA-embedde d tissu e sections . J . His t Cytochem . 37:813-818 . Caudill . D , Asquith , T.N. . an d Lehman-McKeeman , L.D . (1991 ) Bioc l difference s betwee n o&u-globuli n (ot2u ) an d mous e urinar y (MUP) : explanatio n o fmurin e resistanc e t o develo p hyalin e nephropath y (HDN) . Toxicologist 11:13 7 (Abstract) . Cavaggioni . A. . Sorbi . R.T. , Keen . J.N. . Pappin . D.J.C. . an d Findla y (1987 ) Homolog y betwee n th e pyrazine-bindin g protei n fro r mucos a an d majo rurinar y proteins . FEB S Lett . 212:225-22 8 Cavaggioni . A . Findlay . J B.C. , an d Tirindelli . R . (1990 ) Ligan d characteristic s ot homologou s rat an d mous e urinar y prote i pyrazine-bindin g protei n o fcalf . Comp . Biochem . Physiol . 9 6 520 . Charbonneau . M. . Lock . E.A. . Strasser , J. , Cox . M.G. , Turner . M Bus . J.S . (1987 ) 2.2,4-Trimethylpentane-induce d nephrotox i Metaboli c dispositio n o fTM P i n mal e an d femal e Fische r3 ' Toxicol . Appl . Pharmacol . 91:171-181 . Charbonneau . M. . Strasser . J. , Borghoff , S.J. , an d Swenberg . J. A I n vitro hydrolysi s o

f[UCJ-OL-globuli n (a J isolate d fro m n kidney . Toxicologis t 8:13 5 (abstract) . Charbonneau . M. , Strasser . J. . Lock . E . A . Turner . M.J. , an d Swenb J (1989 ) Involvemen t o freversibl e bindin g t o 0L, u -gk>buli n dichlorobenzene-induce d nephrotoxicity . Toxicol . Appl . Ph a 99 : 122-132 . 10 0 10 1

O l o oChatterjee , B. , Hopkins . J , Dutchak , D. , an d Roy . A.K . (1979 ) Superinductio n

o fcL^-globuli n b y actinomyci n D : evidenc e fo rdrug-mediate d increas e i n as mRNA . Proc . Natl . Acad . Sci . 76:1833-1837 . Chatterjee . B. . Demyan , W.F. , Song , C.S. , Garg , B.D. . an d Roy . A.K . (1989 ) Loss o fandrogeni c inductio n o fOL-globuli n gen e famil y i n th e live ro f NI H Blac k

rats . Endocrinolog y 125:1385-1388 . Churchill . D.N. . Fine , A , an d Gault , M.H . (1983 ) Associatio n betwee n hydrocarbo n exposur e an d glomerulonephritis . A n appraisa l o fth e evidence . Nephro n 33:169-172 . Cohen , S.M. , an d Eltwein , L.B . (1990 ) Cel l proliferatio n i n carcinogenesis . Scienc e 249:1007-1011 . Crisp . A J. . Bhalla . A.K. . an d Hoffbrand , B.I . (1979 ) Acut e tubula rnecrosi s afte rexposure t o diese l oil . Brit . Med . J . 2:177 . Cunningham , ML , Foley , J. , Maronpot , R.R. , an d Matthews , H.B . (1991 ) Correlatio n o

fhepatocellula rproliferatio n wit h hepatocarcinogenicity induce d by th e mutageni c noncarcinogen:carcinoge n pai r- 2,6 - an d 2,4-diaminotoluene . Toxicol . Appl . Pharmacol . 107:562-567 . Daniell , W.E. , Couser , W.G. , an d Rosenstock , L . (1988 ) Occupationa l solven t exposur e an d glomerulonephritis . A cas e repor t an d reviewo

f th e literature . J . Am . Med . Assoc . 259:2280-2283 . Daugherty , W.C. , Smith , J.H.Hinz , J.P. . an d Biles , R.W . (1990 ) Subchroni c toxicit y evaluatio n o

ftridecy l acetat e i n rats . Fundam . Appl . Toxicol . 14 : 104-112 . Dayal , H , an d Kinman , J . (1983 ) Epidemiolog y o fkidney cancer . Semin . Oncol . 10:366-377 . Deal , F.H. , Richardson , F.C. . an d Swenberg , J.A . (1989 ) Dos e respons e o f hepatocyt e replicatio n i n rat s followin g continuou s exposur e t o diethylnitrosamin e Cance rRes . 49:6985-6988 . Dees . J.H. , Heatfield , B.M. , Reuber , M.D. , an d Trump , B.F . (1980a ) Adenocarcinom a o fth e kidney . III . Histogenesi s ofrena l adenocarcinoma s i n rat s by N-(4 , -ftuoro-4-biphenytyl)acetamide . J . Natl . Cance rInst . 64:1537-1545 . Dees , J.H. , Heatfield . B.M. . an d Trump , B.F . (1980b ) Adenocarcinom a o f th e kidney . IV . Electronmicroscopi c study o fth e developmen t o

frena l adenocarcinoma s induce d i n rats b y N-(4'- fluoro-4-biphenylyl)acetamide . J . Natl . Cance rInst . 64:1547-1562 . Dekant , W. , Vamvakas , S. , an d Anders , M.W . (1989 ) Bioactivalio n o f nephrotoxi c haloalkane s b y glutathion e conjugatio n —formatio n o f toxi c an d mutageni c intermediate s b y cystein e conjugate B-lyase . Dru g Metab . Rev . 20 : 43-83 . Devesa . S.S. , Silverman , D.T. , McLaughlin , J.K. , Brown , C.C. , Connelly , R.R. , an d Fraumeni , J.F . (1990 ) Compariso n o fth e descriptive epidemiolog y o

furinar y trac t cancers . Cance rCause s an d Contro l 1 : 133-141 . Dice . J.F . (1987 ) Molecula rdeterminant s o fprotei n half-live s i n eukaryoti c cells . FASE B J 1 : 349-357 . Dieppe , PA . Doyle . D.V. , an d Burry , HC . (1978 ) Rena l damag e durin g treatmen t wit h antirheumati c drugs . Brit . Med . J . 2 : 664 .

in o Dietrich , D.R. , an d Swenberg . J.A . (1990 ) Lindan e induce s nephropath y an d rena l accumulatio n o fa2u-globuli n i n mal e bu t no t i n femal e Fische r 34 4 rat s o rmal e NB R rats . Toxicol . Lett . 53:179-181 . Dietrich , DR . an d Swenberg . J.A . (1991a ) NCI-Black-Reite r(NBR ) mal e rat s fai l t o develo p rena l diseas e followin g exposur e t o agent s that induc e ot^-globuli n (OL J nephropathy . Fundam . Appl . Toxicol . 16:749 - 762 . Dietrich , D.R. , an d Swenberg . J.A . (1991b ) Alphas-globuli n i s necessary fo r d-limonen e promotio n ofmal e rat kidne y tumors . Cance rRes . 51:3512 - 3521 . Dodd , D.E. , Losco . P.E. , Troup . CM , Pritts , I.M. . an d Tyler . T.R . (1987 ) Hyalin e droplet nephrosi s i n mal e Fische r34 4 rat s followin g inhalatio n ofdiisobuty l ketone . Toxicol . Indust . Healt h 3:443-457 . Dolan , K.P. , Unterman , R. , McLauglin , M. . Nakhasi , H.L. , Lynch , K.R. , an d Feigelson , P . (1982 ) Th e structure an d expressio n o fver y closel y relate d member s o

fth e OL-globuli n gen e family . J . Biol . Chem . 257 : 13527-13534 . Dominick , M.A. , Robertson , D.G. . Sifler , M.R. . Sustck , R.L. . an d Bobrowski , W.F . (1990 ) Absenc e o fnephrocarcinogenesi s i n mal e rat s administere d gabapentin . a compoun d tha t cause s atpha-2u-globuli n nephropathy . Proceeding s o

fth e America n Colleg e o fVet e rinar

y Pathology . Decembe r 9-14,1990 , p . 4 5 (abstract) . Drayna . D.T. , McLean , J.W. . Wion , K.L. . Trent , J.M. , Drabkin , HA , an d Lawn . R.M . (1987 ) Huma n apolipoprotei n D gene : gen e sequence , chromosom e localization , an d homolog y t o th e ot^-globuli n superfamily . DN A 6:199-228 . Duh , R-W. , an d Asal , N.R . (1984 ) Mortalit y amon g laundr y an d dry cleanin g worker s i n Oklahoma . Am . J . Publi c Healt h 74:1278-1280 . Ekstrom , R.C . (1983 ) Characterizatio n an d metabolis m o fOL-globuli n - a mal e sex-dependen t protei n o fth e rat . Ph.D . thesis . Universit y o f Wisconsin-Madison . Ekstrom , B. , an d Berggard , I . (1977 ) Huma n a,-microglobulin : purificatio n procedure , chemica l an d physicochemica l properties . J . Biol . Che m 252:8048-8057 . Elcombe . C.R. , Rose , M.S. , an d Pratt , IS . (1985 ) Biochemical , histological , an d ultrastructura l change s i n ra t an d mous e live rfollowin g th e administratio n o

t trichloroethylene : possibl e relevanc e t o specie s difference s i n hepatocarcinogenicity . Toxicol . Appl . Pharmacol . 7 9 365-376 . Enterline , P.E. , an d Viren , J . (1985 ) Epidemiologi c evidenc e fo ra n associatio n betwee n gasolin e an d kidne y cancer . Environ . Healt h Persp . 62:303-312 . Evans , GO , an d Morgan , R.J.I . (1986 ) Urinary enzym e measurement s i r mal e rat s afte rora l administratio n o fdecalin . Huma n Toxicol . 5:12 0 (abstract) . Evans , GO. . Goodwin . DA , Parsons , C.E. , an d Read . N.G . (1988 ) Th e effect s o flevamisol e o n urinar

y enzym e measurement s an d proxim a tubul e cel l inclusion s i n mal e rats . Brit . J . Exp . Pathol . 69 : 301-308 .Fan , C.Y. , Butler , W.H. . an d O'Connor . P.J . (1989 ) Cel l an d tissu e specifi c

localizatio n o

fO'-methylguanin e i n th e DN A o frat s give n N nrtrosodimethylamine : effect s o fprotei n deficien t an d norma l diets . Carcinogenesi s 10:1967-1970 . Farber , E . (1988 ) Cance rdevelopmen t an d its natura l history . A cance r preventio n perspective . Cance r62:1676-1679 . Feigelson , P. . an d Kurtz . D.T . (1977 ) Hormona l modulatio n o fspecifi c messenge rRN A specie s i n norma l an d neoplasti c ra t liver . Adv . Enzymol . 47 : 275-312 . Flamm , W.G. . an d Lehman-McKeeman . L.D . (1991 ) Th e huma n relevanc e o fth e rena l tumor-inducin g potentia l o fd-limonen e i n mal e rats : implication s fo rris k

assessment . Regulatory Toxicol . Pharmacol . 13 : 70-86 . Fowlie , AJ.Grasso . P. . an d Bridges . J . W . (1987 ) Rena l an d hepati c lesion s induce d b

y 2.2,4-trimethylpentane . J . Appl . Toxicol . 7:335-341 . Galloway , S.M. , Armstrong . M.J. . Reuben , C . Colman , S. . Brown . B. , Cannon . C. . Bloom , A.D. , Nakamura , F. . Ahmed , M. . Duk , S. . Rimpo , J. . Margolin , B.H. . Resnick . M.A. . Anderson . B. . an d Zeiger , E . (1987a ) Chromosom e aberration s an d siste rchromati d exchange s i n Chines e hamste rovar

y cells : evaluation s o f10 8 chemicals . Environ . Mol . Mutagen . 10(Suppl . 10) : 1-175 . Galloway . S.M. . Deasy . DA. . Bean . C.L. . Kraynak , A.R. , Armstrong , M.J. . an d Bradley . MO . (1987b ) Effect s o fhig h osmoti c strengt h o n chromosom e aberrations , sister-chromati d exchange s an d DN A stran d breaks , an d th e relatio n t o toxicity . Mutat . Res . 189:15-25 . Garg . B.D. , Olson . M.J. . Li . L.C , Mandni . M.A. , an d Roy , A.K . (1987 ) Immunoelectro n microscopi c localizatio n o fOL-globuli n i n th e mal e ra t kidney afte rgasolin e exposure . In : Bailey , G.W . (ed) . Proceeding s o f th e 45t h Annua l Meetin g o fth e Electro n Microscopi c Societ y o fAmerica . Sa n Francisco , California , pp . 872-873 . Garg . BD , Olson . M.J. . Demyan . W.F. . an d Roy . A.K . (1988 ) Rapi d post exposur e deca y o

frx^-globuli n an d hyalin e droplet s i n th e kidney s o f gasoline-treate d mal e rats . J . Toxicol . Env . Healt h 24:145-160 . Garg . B.D. , Olson . M.J. , an d Li , L. C (1989a ) Phagolysosoma l alteration s induce d b

y unleade d gasolin e i n epithelalcellso fth e proxima l convolute d tubule s o

fmal e rats : effect o

fdos e an d treatmen t duration . J . Toxicol . Env . Healt h 26:101-118 . Garg , BD , Olson . M.J. . Li . L.C , Mancini , M.A. . an d Roy , A.K . (1989b ) Estradio l pretreatmen t o fmal e rat s inhibit s gasoline-induce d rena l hyalin e drople t an d alpha- 2u -globuli n accumulation . Researc h Publication , Genera l Motors Researc h Laboratories . GMR-6557 . Marc h 1989 . Gaworski . OL. , MacEwen . J.D. . Vernot , E.H. , Bruner , R.H. , an d Cowan , M.J . (1984 ) Compariso n o fth e subchroni c inhalatio n toxicit y o fpetroleu m an d oi l shal e JP- 5 je t fuels . In : Mehlman , M.A. , Hemstreet , G.P. , Thorpe , J.J. , an d Weaver , N.K . (eds.) , Advance s i n Moder n Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers . Inc. . Princeton , p p 3 - 47 .

10 4 Gaworski . C.L. , MacEwen . J.D. , Vernot , E.H. . Haun . C.C. . Leahy . H.F. , Bruner,R.H..Hall,A..Latendresse.JR. , Mattie.DR..an d PHts.L.L . (1985a ) Evaluatio n of90-da y inhalatio n toxicit y o fpetroleu m an d oi l shal e diese l fue l marin e (DFM) . Harr y G . Armstron g Aerospac e Medica l Researc h Laboratory . AMRL-TR-85-074 . NMRI85-57 , Decembe r1985 . Gaworski . C.L. . Haun , C.C. . MacEwen , J.D. , Vernot , E.H. . Bruner , R.H. . Amster , R.L. . an d Cowan , M.J . (1985b ) A 90-da y vapo rinhalatio n toxicit y stud y o

fdecalin . Fundam . Appl . Toxicol . 5:785-793 . Geertzen.H G M .vandenOuderaa. F J G,andKassenaar,A.A.H . (1973 ) Isolatio n an d metabolis m ot mat e sex-dependen t urinary protei n fro m rats . Act a Endocrinologic a 72:197-208 . Godovac-Zimmermann . J . (1988 ) Th e structura l moti fo fB-lactogtobuli n an d retinol-bindin g protein : a basi c framewor k fo rbindin g an d transpor t o f smal l hydrophobi c molecules ? 1988 . Trend s Biochem . Sci . 13:64-66 . Goldsworthy . T.L. , an d Poop , J.A . (1987 ) Chlorinate d hydrocarbon-induce d peroxisoma l enzym e activit y i n relatio n t o specie s an d orga n carcinogenicity . Toxicol . Appl . Pharmacol . 88:225-233 . Goldsworthy . T.L. . Lyght . O. , Burnett . V.L. . an d Poop , J.A . (1988a ) Potentia l rol e ofo2u-globulin . protei n droplet accumulation , an d ce O replicatio n i n th e renalcarcinogenicityofrats expose d t o trichloroethylene , perchloroethylene , an d pentachloroethane . Toxicol . Appl . Pharmacol . 96 : 367-379 . Goldsworthy . T.L. , Smith-Oliver , T.,Loury , D.J. , Popp . J.A.andButterworth , B.E . (1988b ) Assessmen t o fchlorinate d hydrocarbon-induce d genotoxicit

y an d cel l replicatio n i n ra t kidney cells . Environ . Mol . Mutagen . 1 1 (Suppl . 11) : 39 . Goodman , D.G. , Ward . J.M. , Squire . R.A. . Paxton . M.B. . Reichardt , W.D. , Chu , K.C. , an d Linhart . M.S . (1980 ) Neoplasti c an d non-neoplasti c lesion s i n agin g Osborne-Mende l rats . Toxicol . Appl . Pharmacol . 55 : 433-447 . Goodman , M.T. , Morgenstem , H. , an d Wynder , E.L . (1986 ) A case-contro l stud y o ffactor s affectin g th e developmen t o frena l cel l cancer . Am . J . Epidemiol . 124:926-941 . Gray . J.E . (1986 ) Chroni c progressive nephrosis , rat . In : Jones , T.C.Mohr , U.,an d Hunt , R.D.(eds.) , Urinar y System . (LS I Monograph s o n Patholog y o fLaborator

y Animals . Springer-Vertag . Ne wYork . pp . 174-179 . Green , T . Odum . J. , Nash , J.A. , an d Foster , J.R . (1990 ) Perchtoroethylene induce d rat kidney tumors : a n investigatio n o fth e mechanism s involve d an d thei rrelevanc e t o humans . Toxicol . Appl . Pharmacol . 102:77-89 . Gnsharn , J.W. , Kaufmann , W.K.andKaufmann , D.G . (1983 ) Th e cel l cycl e an d chemica l carcinogenesis . Survey Synth . Pathol . Res . 1:49-66 . Haider , C.A. , Warne . T.M. , Hatoum . N.S . (1984 ) Rena l toxicity o fgasolin e an d relate d petroleu m naphtha s i n mal e rats . In : Mehlman , M.A. , Hemstreet . G.P. . Thorpe . J.J. , an d Weaver , N.K . (eds) , Advance s i n Mode m Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton . New Jersey , pp . 73-88 . Haider . C.A. . Holdsworth . C.E. , Cockrell . BY. , an d Piccirillo , V.J . (1985 ) Hydrocarbo n nephropath y i n mal e rats : identificatio n o fth e nephrotoxi c component s o

funleade d gasoline . Toxicol . Indust . Healt h 1:67-87 .

10 5Hanis . N.M. , Holmes . T.M. . Shallenberger . L.G. , an d Jones , K.E . (1982 )

Epidemiologi c stud yo

frefiner yan d chemica l plan t workers . J . Occup . Med . 24 : 203-212 . Hard , G.C . (1984 ) Hig h frequency , single-dos e mode l o frena l adenoma

/ carcinom a inductio n usin g dimethylnitrosamin e i n Cri:(W)B R rats . Carcinogenesi s 5:1047-1050 . Hard , G. C (1987 ) Chemicall yinduce d epithelia l tumors an d carcinogenesi s o fth e rena l parenchyma . In : Bach , P.H. , an d Lock , E.A . (eds. ) Nephrotoxicit

yi n th e Experimenta l an d th e Clinica l Situation , Par t I . Martinu s Nijhof

fPublishers , Lancaster , pp . 211-250 . Hard . G.C . (1990 ) Tumour s o fth e kidney , rena l pelvi s an d ureter . In : Turusov , V.S. . an d Mohr , U . (eds.) , Patholog yo fTumour s i n Laborator y Animals . Vol . 1 - Tumour s ofth e Rat . Secon d Edition . Internationa l Agenc yfo rResearc h o n Cancer , Lyon . IAR C Scientifi c Publication s No . 99 , pp . 301-344 . Hard . G.C , an d Butler , W.H . (1971 ) Morphogenesi s o fepithelia l neoplasm s induce d i n th e rat kidneyb ydimethylnitrosamine . Cance rRes . 31 : 1496-1505 . Hard , G.C , an d Snowden , R.T . (1991 ) Hyalin e droplet accumulatio n i n roden t kidney proxima l tubules : a n associatio n wit h histiocyti c sarcoma . Toxicol . Pathol , (i n press) . Hard , G.C , Mackay . R.L. , an d Kochhar . O.S . (1984 ) Electro n microscopi c determinatio n ofth e sequenc e o facut e tubula ran d vascula rinjur y induce d i n th e rat kidneyby a carcinogeni c dos e o fdimethylnitrosamine . Lab . Invest . 50:659-672 . Hard , G.C . Alden , C.L. , Stula , E F. , an d Trump . B.F . (1991 ) Proliferative lesion s o

fth e kidneyi n rats . Societ yo fToxicologi c Pathologists . Standar d Syste m o

fNomenclature an d Diagnosti c Pathology . I n press . Harrington.J.M. , Whitby . H. . Gray . C.N. . Reid , F.J. , Aw . T.C . andWaterhouse , J.A . (1989 ) Rena l diseas e an d occupationa l exposur e t o organi c solvents : a case-referen t approach . Brit . J . Industr . Med . 46:643-650 . Haseman , J.K. , Huff , J. . an d Boorman , G.A . (1984 ) Us e o fhistorica l contro l dat a i n carcinogenicitystudie s i n rodents . Toxicol . Pathol . 12:126-135 . Hastie , N.D. , Held , W.A. , an d Toole, J.J . (1979 ) Multipl e gene s codin g fo r th e androgen-regulate d majo rurinar yprotein s o

fth e mouse . Cel l 17 : 449-457 . Haworth , S. , Lawtor , T. , Mortelmans , K. . Speck , W. , an d Zeiger , E . (1983 ) Salmonell a mutagenicitytes t result s fo r25 0 chemicals . Environ . Mutagen . 5 (Suppl . 1) : 1-14 2 Healt h Effects Institute (HEI) . (1985 ) Gasolin e Vapo rExposure an d Huma n Cancer : Evaluatio n ofExistin g Scientifi c Informatio n an d Recommendation s fo rFurthe rResearch . Healt h Effect s Institute , Repor t o fth e Institute' s Healt h ReviewCommittee , Septembe r1985 . Healt h Eflect s Institute (HEI) . (1988 ) A n Updat e o n Gasolin e Vapo r Exposur e an d Huma n Cancer : A n Evaluatio n o fScientifi c Informatio n Publishe d Betwee n 198 5 an d 1987 . Healt h Effect s Institute , Repor t o f th e Institute' s Healt h Revie wCommittee , January6,1988 . Hellsten , S. , Berge , T , an d Wehlin . L. (1981 ) Unrecognise d rena l cel l carcinoma . Clinica l an d pathologica l aspects . Scand . J . Urol . Nephrol . 8 : 273-278 . Higginson , J . Muir . C.S. , an d Buf fler , PA . (1984 ) Th e epidemiolog yo frena l carcinom a i n human s wit h a not e o n th e effect o fexposure to gasoline . In : Mehlman , M.A. , Hemstreet . G.P. . Thorpe . J.J. , an d Weaver , N.K . (eds.) . Advance s i n Moder n Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton . Ne wJersey , pp . 203-226 . Hobson , D.W. , Mattie , D.R. , Bruner , R.H. . Uddin , D.E. , Eurell , T. , an d Olson . C.T . (1986 ) Effec t o fcastratio n o n th e developmen t o f2,2.4 - trimethylpentane-induce d hydrocarbo n nephropath yi n th e mal e F-34 4 rat . Toxicologist 6:17 3 (abstract) . Hoel , D.G. , Haseman , J.K. , Hogan , M.D. , Huff , J . an d McConnell , E.E . (1988 ) Th e impac

t o ftoxicit yo n carcinogenicitystudies , implication s fo r ris k assessment . Carcinogenesi s 9 : 2045-2052 . Hughson , M.D. . Buchwold , D. . an d Fox , M . (1986 ) Rena l neoplasi a an d acquire d cysti c kidne

ydiseas e i n patient s receivin g lon g ter m dialysis . Arch . Pathol . Lab . Med . 110 : 592-600 . Jackson , M.R . (1974 ) Th e natur e o fdimethylnitrosamin e induce d enlargemen t o

frat hepatocyt e nuclei . J . Pathol . 113:197-208 . Jedrzejewski , K. , an d Kugler , P . (1982 ) Peptidase s i n th e kidneyan d urin e o frat s afte rcastration . Histochemistr y74:63-84 . Johnson , D.E . (1987 ) Protei n induce d nephropath yi n mal e rats . In : Hotcenberg , J.S. , an d Winkelhake , J.L . (eds) , Th e Pharmacolog yan d Toxicolog yo

fProteins . Ala n R . Lis s Inc. , Ne wYork , pp.165-171 . Kadamani . S. . Asal . N.R. , andNelson . R.Y . (1989 ) Occupationa l hydrocarbo n exposure an d ris k

o frena l cel l carcinoma . Am . J . Industr . Healt h 15 : 131-141 . Kanerva . R.L. . McCracken , M.S. , Alden . C.L. , an d Stone . L. C (1987a ) Morphogenesi s o

fdecalin-induce d rena l alteration s i n th e mal e rat . Fd . Chem . Toxicol . 25 : 53-61 . Kanerva , R.L. , Ridder . G.M. , Lefever . F.R. , an d Alden . C.L . (1987b ) Compariso n o

fshort-ter m rena l effect s du e t o ora l administratio n o f decali n o rd-limonen e i n youn g adul t mal e Fischer-34 4 rats . Fd . Chem . Toxicol . 25:345-353 . Kanerva , R.L. . Ridder , G.M. , Stone , L.C , an d Alden . C.L . (1987c ) Characterizatio n o

fspontaneou s an d decalin-induce d hyalin e droplets i n kidney s o

fadul t mal e rats . Fd . Chem . Toxicol . 25 : 63-82 . Katz , R.M. . an d Jowett , D (1981 ) Femal e laundr yan d dr y cleanin g workers i n Wisconsin : a mortalit yanalysis . Am . J . Publi c Healt h 71:305-307 . Katz . G.V. . Renner , E.R. , an d Terharr , C.J . (1986 ) Subchroni c inhalatio n toxicit yo fmethy l isoamy l keton e i n rats . Fundam . Appl . Toxicol . 76 : 498-505 . Kaysen , G.A. , Myers . B.D. . Couser . W.G. , Rabkin . R. , an d Felts . J.M . (1986 ) Biolog yo

fDisease . Mechanism s an d consequence s o fproteinuria . Lab . Invest . 54:479-498 . Killeen , J.C. . Wilson , N.H. . Ford . W.H. . Siou . G. , Busey . W.M. , an d Eilrich , G.L . (1990 ) Progressio n o frena l an d forestomac h effects followin g administratio n o

fchlorothaloni l t o rats . Toxicologist 10:26 9 (abstract) . Kimura.H.Odani.S. . Suzuki . J-I.Arakawa.M . an d Ono.T . (1989 ) Kidne y fatt yacid-bindin g protein : identificatio n a s a 2u -globulin . FEB S Letters 246 : 101-104 .Kloss , M.W. . Cox , M.G. , Norton , R.M. , Swenberg . J.A , an d Bus , J.A . (1985) . Sex-dependen t difference s i n th e dispositio n o f( u C-5]2,2,4 - trimethylpentan e i n Fische r34 4 rats . In : Bach , P.H. , an d Lock , E.A . (eds) , Rena l Heterogeneit y an d Target Cel l Toxicity . Wiley , Chichester , pp . 489-492 . Kluwe , W.M , Abdo , KM , an d Huff . J . (1984 ) Chroni c kidney diseas e an d organi c chemica l exposures : evaluation s o fcausa l relationship s i n human s an d experimenta l animals . Fundam . Appl . Toxicol . 4:889-901 . Kugler , P. , an d Vornberger , G . (1986 ) Rena l cathepsin- B activitie s i n ratsafte rcastratio n an d treatmen t wit h se xhormones . Histochemistr y 85 : 157-161 . Kuna , R.A. , an d Ulrich , C.E . (1984 ) Subchroni c inhalatio n toxicit y o ftwo moto rfuels . J . Am . Coll . Toxicol . 3:217-229 . Kurtz , D.T. , an d Feigelson , P . (1977 ) Multihormona l inductio n o fhepati c OL-globuli n mRN A a s measure d b y hybridizatio n t o complementary DNA . Proc . Natl . Acad . Sci . 74:4791-4795 . LaVecchia.C,Negri,E.,DAvanzo,B,andFranceschi,S.(1990 ) Smokin g an d rena l cel l carcinoma . Cance rRes . 50:5231-5233 . Le e K-H , Wells . R.G. , an d Reed . R.R . (1987 ) Isolatio n o fa n olfactory cDNA : similarit y t o retinol-bindin g protei n suggests a rol e i n olfaction . Scienc e 235:1053-1056 . Lehman-McKeeman , L.D. . Rodriguez , PA , Takigiku , R , Caudill , D. , an d Fey , M.L . (1989 ) d-Limonene-induce d mal e rat-specifi c nephrotoxicity : evaluatio n o

fth e associatio n betwee n d-limonen e an d CL^-globulin . Toxicol . Appl . Pharmacol . 99 : 250-259 . Lehman-McKeeman , L.D. , Rivera-Torres , Ml , an d Caudill , D . (1990a ) Lysosoma l degradatio n o fot 2u-globuli n an d a -globulin-xenobioti c conjugates . Toxicol . Appl . Pharmacol . 103:539-548 . Lehman-McKeeman , L.D. , Caudill , D , Takigiku , R , Schneider , RE , an d Young , J.A . (1990b ) Comparativ e dispositio n o fd-limonen e i n rat s an d mice : relevanc e t o male-rat-specifi c nephrotoxicity . Toxicol . Lett . 53 : 193-195 . Lehman-McKeeman , L.D. , Rodriguez , PA , Caudill , D , Fey , M.L. . Eddy , C.L , an d Asquith , T.N . (1991 ) Hyalin e droplet nephropath y resultin g fro m exposure t o 3.5,5-trimethylhexanoyloxybenzen e sulfonate . Toxicol . Appl . Pharmacol . 107 : 429-438 . Lipsky , MM. , an d Trump , B.F . (1988 ) Chemicall y induce d rena l epithelia l neoplasi a i n experimenta l animals . Intl . Rev . Exptl . Pathol . 30:357-383 . Lock , EA , Charbonneau , M , Strasser , J , Swenberg , J.A , an d Bus , J.S . (1987a ) 2,2.4-Trimethylpentane-induce d nephrotoxicity . II . Th e reversibl e bindin g o

fa TM P metabolite t o a rena l protei n fractio n containin g a^-globulin . Toxicol . Appl . Pharmacol . 91:182-192 . Lock , EA , Stonard , MD , an d Elcombe . C.R . (1987b ) Th e inductio n o f [omega ] an d 0-oxidatio n o ffatty acid s an d effec

t o n O L globuli n conten t i n th e live ran d kidney o frat s administere d 2,2,4-trimethytpentane . Xenobiotic a 17:513-522 . Logothetopoulos , J , an d Weinbren , K . (1955 ) Naturall y occurrin g protei n droplet s i n th e proxima l tubul e o fth e rat' s kidney . Brit . J . Exptl . Pathol . 36 : 402-406 . 10 8 Loury . D.J , Smith-Oliver , T , an d Butterworth , BE . (1987 ) Assessmen t o f unschedule d an d replicativ e DN A synthesi s i n rat kidney cell s expose d iQ_yjti Q o rinviv Q to unleade d gasoline . Toxicol . Appl . Pharmacol . 87 : 127-140 . Lynge , E , an d Thygesen , L. (1990 ) Primary live rcance ramon g wome n i n laundr y an d dry-cleanin g wor k i n Denmark . Scand . J . Wor k Enviro n Healt h 16:108-112 . Maack , T , Park , OH , an d Camargo , M J.F . (1985 ) Rena l filtratio n transport , an d metabolis m o fproteins . In : SekJin , D.W , an d Giebisch . G . (eds.) , Th e Kidney : Physiolog y an d Pathophysiology . Rave n Press . Ne wYork , pp . 1773-1803 . MacFarfand , H.N . (1984 ) Xenobioti c induce d kidne y lesions : hydrocarbons . Th e 90-da y an d 2-yea rgasolin e studies . In : Mehlman . M . A , Hemstreet . G.P , Thorpe , J.J , an d Weaver . N.K . (eds.) . Advance s i n Mode m Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton , Ne w Jersey , pp . 51-56 . MacFarfand . H.N , Ulrich . C.E , HokJsworth , C.E , Kitchen , D.N , Hallrwell , W.H. , an d Blum . S O (1984 ) A chroni c inhalatio n stud y wit h unleade d gasolin e vapor . J . Am . Coll . Toxicol . 3:231-248 . Maclnnes , J.I , Nozik , E.S , an d Kurtz , D.T . (1986 ) Tissue-specifi c expressio n o fth e rat alpha , globuli n gen e family . Molec . Cell . Biol . 6:3563-3567 . MacNaughton , M.G" . an d Uddin , D E (1984 ) Toxicolog y o fmixe d distillate an d high-energ y syntheti c fuels . In : Mehlman , MA , Hemstreet . G.P , Thorpe . J.J , an d Weaver . N.K . (eds) , Advance s i n Moder n Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton , Ne w Jersey , pp . 121-132 . Mancini , MA , Majumdar , D , Chatterjee . B , an d Roy . A.K . (1989 ) Alpha,, , globuli n i n modifie d sebaceou s gland s wit h pheromona l functions : localizatio n o

fth e protei n an d its mRN A i n preputial , Meibomian , an d periana l glands . J . Histochem . Cytochem . 37:149-157 . Masoro , E. J , an d Yu , B.P . (1989 ) Die t an d nephropathy . Lab . Invest . 60 : 165-167 . Mattie , D.R. , Bruner , R.H , Hoeflich . T.J , an d Kendrick , J.M . (1988 ) Effects o fdecali n an d JP-1 0 o n th e l u ne t io n an d morpholog y o fmal e ra

t kidneys . Toxicologist 8:18 7 (abstract) . Maunsbach , A B (1966a ) Observation s o n th e segmentatio n o fth e proxima l tubul e i n th e rat kidney . Compariso n o fresult s fro m phas e contrast , fluorescenc e an d electro n microscopy . J . Ultrastruct . Res . 16 : 239-258 . Maunsbach , A.B . (1966b ) Electro n microscopi c observation s o fcytoplasmi c bodie s wit h crystallin e pattern s i n ra t kidne y proxima l tubul e cells . J . Ultrastruct . Res . 14:167-189 . McCredie , M , Ford , J.M , an d Stewart . J.H . (1988 ) Ris k factors fo rcance r o fth e rena l parenchyma . Int . J . Cance r42:13-16 . McGregor , D.B , Brown , A , Cattanach . P , Edwards , IA , McBride , D , Riach , C. . Caspary , W. J . (1988 ) Response s o fth e L5178 Yt k

/t k mouse lymphom a cel l forwar d mutatio n assay . III . 7 2 code d chemicals . Environ . Mo l Mutagen . 12 ; 85-154 . 10 9McLaughlin , J.K . (1984 ) Ris k factors fro m a population-base d case-contro l stud y o frena l cancer . In : Mehlman , M . A , Hemstreet , G.P , Thorpe , J.J , an d Weaver . N K

. (eds.) , Advances i n Mode m Environmenta l Toxicology . Vol . VII . Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers , Inc. , Princeton . Ne wJersey , pp . 227-244 . McLaughlin , J.K , an d Schuman , L.M . (1983 ) Epidemiolog y o frena l cel l carcinoma . Rev . Cance rEpidemiol . 2:170-210 . McLaughlin , J.K , Mandel , J.S , Blot , W.J , Schuman , L.M , Mehl . E.S , an d Fraumeni , J.F . (1984 ) A population-base d case-contro l stud y o frena l cel l carcinoma . J . Natl . Cance rInst . 72:275-284 . McLaughlin , J.K , Blot . W.J , Mehl . E.S , Stewart . PA , Venable . F.S , an d Fraumeni , J.F . (1985 ) Petroleum-relate d employmen t an d rena l cel l cancer . J . Occup . Med . 27 : 672-674 . McLaughlin , J.K , Malker , HSR , Stone . B.J , Weiner . J.A , Maker . B.K. , Ericsson , J.L.E , Blot . W.J , an d Fraumeni . J.F . (1987 ) Occupationa l risk s fo rrena l cance ri n Sweden . Brit . J . Industr . Med . 44:119-123 . Mortelmans . K , Haworth , S , Lawlor . T , Speck , W , Tainer , B , an d Zeiger , E . (1986 ) Salmonell a mutagenicit y tests . II . Result s fro m th e testin g o f 27 0 chemicals . Environ . Mutagen . 8 (Suppl . 7) : 1-119 . Motwani , N.M , Caron , D , Demuan , W.F , Chatterjee . B , Hunter , S , Poulik . M D , an d Roy . A.K . (1984 ) Monoclona l antibodie s t o a^-globuli n an d immunocytofluorometri c analysi s ofa -globulin-synthesizin g hepatocyte s durin g androgeni c inductio n an a aging . J . Biol . Chem . 259 : 3653-3657 . Muggia , F.M , Heinemann , HO , Farhangi , M , Osserman , E.F . (1969 ) Lysozymuri a an d rena l tubula rdysfunctio n i n monocyti c an d myelomonocyti c leukemia . Am . J . Med . 47:351-366 . Murty . C.V.R , Sarkar . F.H , Mancini , MA , an d Roy , A.K . (1987 ) Sexindependen t synthesi s o fcx^-globuli n an d it

s messenge rribonuclei c aci d i n th e ra t preputia l gland : biochemica l an d immunocytochemica l analysis . Endocrinolog y 121:1000-1005 . Murty , C.V.R , Olson , M.J , Garg , BD , an d Roy , A.K . (1988 ) Hydrocarbon induce d hyalin e droplet nephropath y i n mal e rat s durin g senescence . Toxicol . Appl . Pharmacol . 96:380-392 . Nationa l Cance rInstitute (NCI ) (1978a ) Bioassay o fchlorothaloni l fo r possibl e carcinogenicity . Carcinogenesi s Testin g Program , Nationa l Cance rInstitute . DHE WPublicatio n No . (NIH ) 78-84 1 (NTP-TR-41) . Nationa l Cance rInstitut e (NCI ) (1978b ) Bioassay o fhexachloroethan e fo r possibl e carcinogenicity . Carcinogenesi s Testin g Program , Nationa l Cance rInstitute . CA S No . 67-72-1 . DHE WPublicatio n No . (NIH ) 78 - 1318 . (NTP-TR-68) . Nationa l Cance rInstitute (NCI ) (1987 ) Annua l Cance rStatistic s Review . Nationa l Cance rInstitute , U S DHHS . NI H Publicatio n No . 88-2789 . Nationa l Toxicolog y Progra m (NTP ) (1983 ) Carcinogenesi s bioassay o f pentachloroethan e (CA S No . 76-0 1 -7 ) i n F344/ N rats an d B6C3F 1 mice (gavag e study) . NTP-81-34 . NI H Publicatio n No . 83-1788 . NTP-TR 232 , Apri l 1983 . Nationa l Toxicolog y Progra m (NTP ) (1986a ) Toxicolog y an d carcinogenesi s studies o

fisophoron e (CA S No . 78-59-1 ) i n F344/ N rat s an d B6C3F 1

11 0 mic e (gavag e studies) . NI H Publicatio n No . 86-2547 . NTP-TR-291 . January 1986 . Nationa l Toxicolog y Progra m (NTP ) (1986b ) Toxicolog y an d carcinogenesi s o ftetrachloroethylen e (perchloroethylene ) (CA S No . 127-18-4 ) i n F344 / N rat s an d B6C3F 1 mice (inhalatio n studies) . NI H Publicatio n No . 8 6 2567 . NTP-TR-311 . Augus t 1986 . Nationa l Toxicolog y Progra m (NTP ) (1987a ) Toxicolog y an d carcinogenesi s studie s o

f1,4-dichlorobenzen e (CA S No . 106-46-7 ) i n F344/ N rat s an d B6C3F 1 mic e (gavag e studies) . NI H Publicatio n No . 87-2575 . NTP TR-319 , Januar

y 1987 . Nationa l Toxicolog y Progra m (NTP ) (1987b ) Toxicolog y an d carcinogenesi s studie s o

fdimethy l methylphosphonat e (CA S No . 756-79-6 ) i n F344/ N rat s an d B6C3F 1 mic e (gavag e studies) . NI H Publicatio n No . 87-2579 . NTP-TR-323 . Novembe r1987 . Nationa l Toxicolog y Progra m (NTP ) (1988 ) Toxicolog y an d carcinogenesi s studie s o

ftrichloroethylen e (CA S No . 79-0 1 -6 ) i n fou rstrain s ofrat s (ACI , August , Marshall , Osborne-Mendel ) (gavag e studies) . NI H Publicatio n No . 88-2529 . NTP-TR-273 , Apri l 1988 . Nationa l Toxicolog y Progra m (NTP ) (1989 ) Toxicolog y an d carcinogenesi s studie s o

fhexachloroethan e (CA S No . 67-72-1 ) i n F344/ N rat s (gavag e studies) . NI H Publicatio n No . 89-2816 . NTP-TR-361 . August 1989 . Nationa l Toxicolog y Progra m (NTP ) (1990 ) Toxicolog y an d carcinogenesi s studie s o

fd-limonen e (CA S No . 5989-27-5 ) i n F344/ N rats an d B6C3F 1 mic e (gavag e studies) . NI H Publicatio n No . 88-2902 . NTP-TR-347 , January 1990 . Nationa l Toxicolog y Progra m (NTP ) (1991a ) Toxicit y studie s of pentachlorobenzen e i n F344/ N rat s an d B6C3F , mice (fee d studies ) (NT P Tox6) . NI H Publicatio n No . 91-3125 , Januar y 1991 . Nationa l Toxicolog y Progra m (NTP ) (1991b ) Toxicity studie s o t 1.2,4,5 - tetrachlorobenzen e i n F344/ N rat s an d B6C3F , mic e (fee d studies ) (NT P To x7) . NI H Publicatio n No . 91-3126 . January 1991 . Neuhaus , O.W , an d Ftory , W . (1978 ) Age-dependen t change s i n th e excretio n o

furinar y protein s by th e rat . Nephro n 22:570-576 . Neuhaus , O.W , an d Lerseth , D.S . (1979 ) Dietary contro l o fth e rena l reabsorptio n an d excretio n ofOL-globulin . Kidney Intl . 16:409-415 . Neuhaus . O.W , Flory , W , Biswas , N , an d Hollerman , C.E . (1981 ) Urinary excretio n o

fa^-globuli n an d albumi n by adul t mal e rat s followin g treatmen t wit h nephrotoxi c agents . Nephro n 28:133-140 . Newsom , G.D , an d Vugrin , D . (1987 ) Etiologi c factors i n rena l cel l adenocarcinoma . Semin . Nephrol . 7:109-116 . Nicoll . J.W , Swann , P.F , an d Pegg , A.E . (1975) i Ef fect o fdimethylnitrosamin e o n persistenc e o

fmethylate d guanine s i n rat live ran d kidney DNA . Natur e 254 : 261-262 . Nogueira , E . (1987 ) Ra t rena l carcinogenesi s afte rchroni c simultaneou s exposure t o lea d acetat e an d N-nitrosodiethylamine . Virchows Arch . (Cel l Pathol. ) 53 : 365-374 . Offic e o fScienc e an d Technolog y Policy (OSTP ) (1985) . Chemica l Carcinogens ; A Reviewo fth e Scienc e an d it s Associate d Principles .

mOffic e o fScienc e an d Technolog yPolicy , Februar y1985 . Federa l Register , Marc h 14.1985 , pp . 10371-10442 . Oliver , J , an d MacDowell , M . (1958 ) Cellula rmechanism s o fprotei n metabolis m i n th e nephron . VII . Th e characteristic s an d significanc e o f th e protei n absorptio n droplets (hyalin e droplets) i n epidemi c hemorrhagi c feve ran d othe rrena l diseases . J . Exptl . Med . 107:731-754 . Olson , M.J , Garg , BD , Murty , C.V.R , an d Roy , A.K . (1987 ) Accumulatio n o fct^-globuli n i n th e rena l proxima l tubule s o fmal e rat s expose d t o unleade d gasoline . Toxicol . Appl . Pharmacol . 90:43-51 . Olson , M.J , Mancini , MA , Garg , BD , an d Roy , A.K . (1988 ) Leupeptin mediate d alteratio n o frena l phagolysosomes : similarityt o hyalin e droplet nephropath yo

fmal e rats expose d to unleade d gasoline . Toxicol . Lett . 41:245-254 . Olson , M.J , Johnson , J.T , an d Rekfy , CA . (1990 ) A compariso n o fmal e ra t an d huma n urinar

yproteins : implication s fo rhuma n resistanc e t o hyalin e droplet nephropathy . Toxicol . Appl . Pharmacol . 102:524-536 . Paganini-Hill . A , Glazer , E , Henderson , BE , an d Ross , R.K . (1980 ) Cause-specifi c mortalityamon g newspape rwe b pressmen . J . Occup . Med . 22:542-544 . Papiz , M.Z , Sawyer , L , Eliopoulos , E.E. . North , ACT , Findlay , J.B.C , Sivaprasadarao , R , Jones , T.A , Newcomer , ME , an d Kraulis , P.J . (1986 ) Th e structure o fB-lactoglobuli n an d its similarityt o plasm a retinol-bindin g protein . Natur e 324:383-385 . Parker . GA.Bogo.V , an d Young , R. W(1981 ) Acut e toxicit y o fconventiona l versu s shale-derive d JP 5 jet fuel : ligh t microscopic , hematologic , an d seru m chemistr

ystudies . Toxicol . Appl . Pharmacol . 57:302-317 . Partanen , T , Heikkiti , P , Hemberg , S , Kauppinen , T , Moneta , G , an d Ojajaarvi , A . (1991 ) Rena l cel l cance ran d occupationa l exposure t o chemica l agents . Scand . J . Wor k Environ . Healt h 17:231-239 . Pegg . A.E . (1984 ) Methylatio n o fth e Oppositio n o

fguanin e i n DN A i s th e mos t latel yinitiatin g even t i n carcinogenesi s bymethylatin g agents . Cance rInvestig . 2:223-231 . Pervaiz , S , an d Brew , K . 1987 . Homolog yan d structure-functio n correlation s betwee n a.-aci d glycoprotei n an d seni m retinol-bindin g protei n an d its relatives . FASE B J . 1:209-214 . Pesce . A.J , Ctyne , D.H , Pollak , V.E , Kant , S.F , Foulkes , E.C , an d Selenke , W.M . (1980 ) Rena l tubula rinteraction s o fproteins . Clin . Biochem . 13:209-215 . Peterson , P . A , an d Berggard , I . (1971 ) Isolatio n an d propertie s o fa huma n retinol-transportin g protein . J . Biol . Chem . 246:25-33 . Pevsner . J , Reed , RR , Feinstein , P.G , an d Snyder , S.H . (1988 ) Molecula r clonin g o

fodorant-bindin g protein : membe ro fa ligan d carrie rfamily . Scienc e 241:336-339 . Phillips , R.D , an d Cockrell , B.Y . (1984 ) Kidneystructura l change s i n rat s followin g inhalatio n exposure to C l0 -C n isoparaf fink : solvent . Toxicolog y 3:261-273 . Phillips , R.D , an d Egan , G.F . (1984 ) Effec t o fC, 0 -C „ isoparaffini c solven t o n kidneyfunctio n i n Fische r34 4 rat s durin g eigh t week s o finhalation . Toxicol . Appl . Pharmacol . 73:500-510 . 11 2 Phillips , R.D , Moran . E.J , Dodd . D.E. . Fowler . EH , Kary . CD , a i O'Donoghue , J . (1987 ) A 14-wee k vapo rinhalatio n toxicit ystudy methy l Isobuty l ketone . Fundam . Appl . Toxicol . 9:380-388 . Phillips . S.C , Petrone , R.L , Hemstreet , G.P . (1988 ) A revie wo l th e n o neoplasti c kidne

y effects o

fhydrocarbo n exposure i n humans . Occup a Med : Stat e o

fth e Ar t Review s 3:495-509 . Pickle , L.W , Mason , T.J , Howard , N , Hoover , R , an d Fraumeni , J . (1987 ) Atla s o

fU.S . cance rmortalit yamon g whites : 1950-19 8 Washington , D.C . U.S . Departmen t o fHealt h an d Huma n Servk > Publicatio n 4 6 (NI H 87-2900) . Pirani , C.L , Sitva , F.G , Appel . G. B (1983 ) Tubuto-interstttia l diseas e multipl e myelom a an d othe rnonrena l neoplasias . In : Cotran , R. J Brenner , B.M , an d Stein , J.H . (eds.) , Tubulo-lnterstitia l Nephropath y Churchil l Livingstone , Ne wYork . pp . 287-334 . Pilot , H. C (1982 ) Th e natura l histor yo fneoplasti c development : t l relatio n o

fexperimenta l model s t o huma n cancer . Cance r49:12 0 1211 . Poole , C , Satterfiekj , M.H , Levin , L , Rothman , K.J , an d Dreyer , N . (1990 ) A case-contro l stud y o fkidne ycance ramon g petroleu m refin e workers . Fina l Repor t to America n Petroleu m Institute , Washingf t D.C . b yEpidemiolog yResource s Inc . Chestnu t Hill , Massachuset t January2nd . 1990 . Pruzanski , W , an d Platts . M.E . (1970 ) Seru m an d urinar y proteins.tysozy n (muramidase) , an d rena l dysfunctio n i n mono - an d myelomonocy t leukemia . J . Clin . Invest . 49:1694-1707 . Read , N.G , Astbury , P.J , Morgan , R.J.I , Parsons , D.N , an d Port , CJ.(19 8 Inductio n an d exacerbatio n o fhyalin e droplet formatio n I n th e proxl m tubula rcell s o

fth e kidneys fro m mal e rat s receivin g a variet y pharmacologica l agents . Toxicolog y52:81-101 . Redmond , C.K. , Ctocco . A. , Lloyd . W , an d Ruch . H. W(1972 ) Long-te r mortalitystud yo

fsteekvorkers . VI . Mortalityfro m malignan t neoplas n amon g cok

e ove n workers . J . Occup . Med . 14:621-629 . Reese , A.J.M. . an d Winstanley . D.P . (1958 ) Th e smal l tumour-lik e leslo r o fth e kidney . Brit . J . Cance r12:507-516 . Richardson , K.A , Wilmer , J.L , Smith-Simpson , D , andSkopek . T.R.(198 ( Assessmen t o

fth e genotoxl c potentia l o funleade d gasolin e an d 2,2 / trimethylpentan e i n huma n lymphoblast s I n vitro . Toxicol . App Pharmacol . 82:316-322 . Richardson , A , Butler , J.A , Rutherford , M.S. , Semsei , I , Gu , M- Z Fernandas . G , an d Chalng . W-H . (1987 ) Effec t o fag e an d dieta r restrictio n o n th e expressio n o fot^-globulin . America n See . Btocher r Mol . Biol . 12821-12825 . Ridder . G . Vo n Bargen , E , Parker . R . Alden , C (1987 ) A comparativ e stud yo fth e acut e effect s o

fd-limonen e an d decali n o n mal e r a nephropathy . Toxicologist 7:23 8 (abstract) . Ridder . GM , Vo n Bargen . EC , Parker . R.D , an d Alden , C.L . (198 8 Spontaneou s an d induce d accumulatio n o fot ^ globuli n i n th e kidne ] cortexo frats an d mice . Toxicologist 8:8 9 (abstract) . 11 3Ridder , GM , Vo n Bargen , EC , Alden . C.L , an d Parker . R.D . (1990 )

Increase d hyalin e drople t formatio n i n mal e rat s expose d t o decali n i s dependen t o n th e presenc e o fa 2 u globulin . Fundam . Appl . Toxicol . 15 : 732-743 . Ritchie , A.W.S , an d Chisholm , GO . (1983 ) Th e natura l histor y o frena l carcinoma . Semin . Oncol . 10:390-400 . Rosanoff.K.A.andSiegel.M. R (1981 ) Mechanis m o factio n an d fat e o

fth e fungicid e chlorothaloni l (2,4,5.6-tetrachloroisophthalonitrile) i n biologica l system s 3 . Interactio n wit h mammalia n DNA , histones . an d isolate d rat live rnuclei . Pesticid e Biochem . Physiol . 16:120-128 . Roy , A.K , an d Neuhaus , O.W . (1967 ) Androgeni c contro l o fa sexdependen t protei n i n th e rat . Natur e 214:618-620 . Roy , A.K , an d Chatterjee , B . (1983 ) Sexua l dimorphis m i n th e liver . Ann . Rev . Physiol . 45:37-50 . Roy , A.K , McMinn . DM , an d Biswas , N.M . (1975 ) Estrogeni c inhibitio n o f th e hepati c synthesi s o fa -globuli n i n th e rat . Endocrinolog y 97:150 1 - 1508 . Roy , A.K , Nath , T.S , Motwani , N.M , an d Chatterjee , B . (1983 ) Age dependen t regulatio n o fth e polymorphi c form s o fa 2 -globulin . J . Biol . Chem . 258:10123-10127 . Savitz , DA , an d Moure , R . (1984 ) Cance rrisk amon g oi l refiner

y workers : a reviewo

fepidemiologica l studies . J . Occup . Med . 26:662-670 . Sawyer , L . (1987 ) Protei n structur e - On e fol d amon g many . Natur e 327 , 659 . Selikoff , I.J , Hammond , E C , an d Seidman , M . (1979 ) Mortalit y experienc e o finsulatio n workers i n th e Unite d State s an d Canada , 1943-76 . Ann . NY . Acad . Sci . 330:91-116 . Serve , MP , Olson . OT , Llewellyn , B.M. . Bruner , R.H , Yu . K.O. , an d Hobson , D.W . (1988 ) Th e metabolis m an d nephrotoxicit y o ftetrali n i n Fische r34 4 rats . Toxicologist 8:18 0 (abstract) . Shapiro , L.E , an d Sachchidananda , J . (1982 ) Regulatio n o fprotein s b

y thyroi d hormon e an d glucocorticoid : th e response s o fhepati c OL globuli n an d pituitar

y growt h hormon e diffe ri n adul t mal e hypothyroi d rats . Endocrinolog y 111 : 653-660 . Short , B.G , Burnett , V.L , an d Swenberg , J.A . (1986 ) Histopatholog y an d cel l proliferatio n induce d b y 2.2,4-trimethylpentan e i n th e mal e ra t kidney . Toxicol . Pathol . 14:194-203 . Short , B.G , Burnett , V.L , Cox , M.G , Bus_J.S , an d Swenberg , J.A . (1987 ) Site-specifi c rena l cytotoxicit y an d cel l proliferatio n i n mal e rats expose d to petroleu m hydrocarbons . Lab . Invest . 57:564-577 . Short , B.G , Burnett , V.L , an d Swenberg , J.A . (1989a ) Elevate d proliferatio n o fproxima l tubul e cell s an d localizatio n o faccumulate d ot^-globuli n i n F34 4 rat s durin g chroni c exposure t o unleade d gasolin e o r2,2,4 - trimethylpentane . Toxicol . Appl . Pharmacol . 101:414-431 . Short , B.G , Steinhagen , W.H , an d Swenberg , J.A . (1989b ) Promotin g effect s o

funleade d gasolin e an d 2,2,4-trimethylpentan e o n th e developmen t o

fatypica l cel l foc i an d rena l tubula rcel l tumors i n rats expose d to N-ethyl-N-hydroxyethylnilrosamine . Cance rRes . 49:6369 - 6378 . Siemiatycki , J , Dewar , R , Nadon , L , Gerin , M , Richardson . L , an d Wacholder.S . (1987 ) Association s betwee n severa l site s o fcance ran d twelve petroleum-derive d liquids . Scand . J . Wor k Environ . Healt h 13 : 493-504 . Sippel , A.E. , Feigelson , P , an d Roy , A.K . (1975 ) Hormona l regulatio n o l th e hepati c messenge rRN A level s fo ra ^ globulin . Biochemistr y 14:825 - 829 . Sippel , A.E. . Kurtz . D.T , Morris , H.P , an d Feigelson . P . (1976 ) Compariso n o fiayjvo.translationa l rate s an d messenge rRN A level s ot OL-globuli n i n ra t live ran d Morri s hepatom a 5123D . Cance rRes . 36:3588-3593 . Smith , AH , Sheam , VI , an d Wood , R.(1989 ) Asbesto s an d kidne y cancer : th e evidenc e support s a causa l association . Am . J . Industr . Med . 16 : 159-166 . Solet , D , an d Robbins . T.6 . (1991 ) Rena l functio n i n dr y cleanin g worker s expose d t o perchloroethylene . Am . J . Industr . Med . (I n press) . Solleveld . HA , Haseman , J.K , an d McConnell , E.E . (1984 ) Natura l histor y o fbod y weigh t gain , survival , an d neoplasi a i n th e F34 4 rat . J . Natl . Cance rIns

t 72 * 929-94 0 Steenland . N.K , Thun . M.J , Ferguson , C.W. , an d Port . F.K . (1990 ) Occupationa l an d othe rexposure s associate d wit h mal e end-stag e rena l disease : a case/contro l study . Am . J . Publi c Healt h 80:153-157 . Stonard . M.D , Foster . J.R , Phillips . P.G.N , Simpson , M.G , Lock , EA . (1985 ) Hyalin e droplet formatio n i n rat kidney induce d by 2,2,4 - trimethylpentane . In : Bach , PH . an d Lock . EA . (eds) . Rena l Heterogeneit

y andTargetCeUToxicity . Joh n Wiley an d Sons , Chichester , pp . 485-488 . Stonard , M.D , Phillips . P.G.N , Foster , J.R , Simpson , M.G , an d Lock . E.A . (1986 ) OL-Globulin : measuremen t i n rat kidney followin g administratio n o f2,2,4-trimethylpentane . Toxicolog y 41:161-168 . Stonard , M.D . (1987 ) Proteins , enzyme s an d cell s i n urin e a s indicators o f th e site o

frena l damage . In : Bach , PH , an d Lock , E.A . (eds) , Nephrotoxicit

y i n th e Experimenta l an d Clinica l Situation . Par t 2 . Martinu s Nijhoff , Lancaster , pp . 563-592 . Stone , L.C , Kanerva , R.L , Burns . J.L , an d Alden , C.L . (1987 ) Decalin induce d nephrotoxicity : light an d electronmicroscopi c examinatio n o f th e effects o

fora l dosin g o n th e developmen t o fkidney lesion s i n th e rat . Fd . Chem . Toxicol . 25:43-52 . Strasser , J , Charbonneau , M , Borghoff . S. J . Turner , M.J , an d Swenberg , J.A . (1988 ) Rena l protei n drople t formatio n i n mal e Fische r34 4 rats afte risophoron e (IPH ) treatment . Toxicologist 8:13 6 (abstract) . Swenberg , J.A . (1989 ) Lecture sponsore d b y Internationa l Life Science s Institute , presente d at Brooking s Institution , Washingto n D.C , Decembe r 13 . Swenberg , J.A . (1991 ) Editorial . Ris k assessmen t o l chemical s causin g a 2o globuli n nephropathy . Reg . Toxicol . Pharmacol . 13:1-2 . Swenberg . J.A , Short , B , Borghoff , S , Strasser . J , an d Charbonneau , M . (1989 ) Th e comparative pathobiolog y o fa^-globuli n nephropathy . Toxicol . Appl . Pharmacol . 97 : 35-46 .Szoka . PR , an d Paigen . K. (1978 ) Regulatio n o fmous e majo rurinar

y protei n productio n b yth e MUP- A gene . Genetic s 90:597-612 . Tannenbaum . M . (1971 ) Ultrastructura l patholog yo fhuma n rena l cel l tumors . Pathol . Ann . 6 : 249-277 . Tannenbaum , M. ( 1983 ) Surgica l an d histopatholog yo frena l tumors . Sem . Oncol . 10 : 385-389 . Thier . R , Peter . H , Wiegand . H.J , an d Bolt , H.M . (1990 ) DN A bindin g stud y o fisophoron e i n rat s an d mice . Arch . Toxicol . 64:684-685 . Thomas . T.L , Decoufle . P , an d Moure-Eraso . R . (1980 ) Mortalityamon g worker s employe d i n petroleu m refinin g an d petrochemica l plants . J . Occup . Med . 22:97-103 . Thomas . F.B , Haider . CA , Holdsworih . C.E , an d Cockrell . B.Y . (1985 ) Hydrocarbo n nephropath yi n mal e rats . Tempora l an d morphologi c characterizatio n o

fth e rena l lesions . In : Bach , PH , an d Lock , E.A . (eds.) . Rena l Heterogeneityan d Targe t Cel l Toxicity . Joh n Wile yan d Sons . Ne wYork . pp . 477-480 . Thorhallson , P. . an d Tulinius . H . (1981 ) Tumor s i n Iceland . 3 . Malignan t tumors o

fkidney . A histologica l classification . Act a Path . Microbiol . Scand . Sect . A 89:403-410 . Thung . P.J . (1962 ) Physiologica l proteinure a i n mice . Act a Physiol . Pharmacol . Neert . 10:248-261 . Trump . B.F , Lipsky . MM , Jones . T.W. , Heatfield . B.M. , Higginson , J , Endicott , K , an d Hess , H.B . (1984a ) A n evaluatio n o fth e significanc e o fexperimenta l hydrocarbo n toxicit yt o man . In : Mehlman , MA , Hemstreet . G.P , Thorpe . J.J , an d Weaver , N.K . (eds.) . Advance s i n Mode m Environmenta l Toxicology . Vol . VII , Rena l Effect s o fPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers . Inc. . Princeton , pp . 273 - 288 . Trump . B.F , Jones , T.W , an d Heatfield . B.M . (1984b ) Th e biolog yo fth e kidney . In : Mehlman . MA , Hemstreet , G.P , Thorpe . J.J , an d Weaver , N.K . (eds.) , Advance s i n Mode m Environmenta l Toxicology , Vol . VII . Rena l Effect s ofPetroleu m Hydrocarbons . Princeto n Scientifi c Publishers . Inc. , Princeton , pp . 27-49 . Tsuji . M , Fujisaki . Y , Arikawa . Y , Masuda . S , Tanaka . T , Sato . K , Noda . K , We , H , an d Kikuchi , M . (1975 ) Studie s o n d-limonene . a s gallston e sotubilizer(IV) . Chroni c toxicit yi n dogs . Pharmacometric s 9:775-808 . (Japanese ) Umemura , T , Takada , K , Ogawa . Y , Kamata , E , Saito . M , an d Kurokawa , Y . (1990) . Se xdifferenc e i n inhalatio n toxicit yo fp-dichlorobenxen e (p DCB ) i n rats . Tox . Lett . 52:209-214 . Universitie s Associate d fo rResearc h an d Educatio n i n Patholog y(UAREP ) (1983 ) Hydrocarbo n toxicity : acute , subchronic , an d chroni c effect s i n relatio n to unleade d gasolin e exposure o frodent s wit h comment s o n th e significanc e to huma n health . Universitie s Associate d fo rResearc h an d Educatio n i n Pathology . Inc . Contract No . PS- 6 UARE P (504-2 ) wit h th e America n Petroleu m Institute . Decembe r1983 . Unterman , R.D , Lynch , K.R , Nakhasi , H.L , Dolan , K.P , Hamilton , J.W , Cohn , D.V. , an d Feigelson , P . (1981 ) Clonin g an d sequenc e o fsevera l OL^-globuli n cDNAs . Proc . Natl . Acad . Sci . U.S.A . 78 , 3478-3482 . 11 6 U S Environmenta l Protectio n Agenc y(USEPA ) (1985 ) Hn^ft h Assessment Documen t fo rTetrachloroethylen e (Perchloroethylene) . Offic e o fHealt h an d Environmenta l Assessment , U.S . Environmenta l Protectio n Agency , Jul y1985 . pp . 8-23 . 8-24 . U S Environmenta l Protectio n Agenc y(USEPA ) (1986 ) Guideline s fo r Carcinoge n Ris k

Assessment . 5 1 F R 33992-34002 . Septembe r24 , 1986 . U S Environmenta l Protectio n Agenc y(USEPA ) (1987 ) Evaluatio n o fth e carcinogenicit

yo funleade d gasoline . Carcinoge n Assessment Group . Unite d State s Environmenta l Protectio n Agency , Septembe r1987 . U S Environmenta l Protectio n Agenc y(USEPA ) (1988 ) Chtorothalonil . Healt h advisory . Office o fDrinkin g Water , U.S . Environmenta l Protectio n Agency . Jun e 1988 . Vamvakas , S , Herkenhoff . M , Dekant . W , an d Henschler , D . (1989 ) Mutagenicit

yo ftetrachloroethen e i n th e Ame s test—metaboli c activatio n byconjugatio n wit h glutathione . J . Biochem . Toxicol . 4:21-27 . Vandoren , G , Mertens , B , Heyns , W , Va n Baelen , H , Rombauts . W , an d Verhoeven . G . (1983 ) Differen t form s o fa^-gtobuli n i n mal e an d femal e rat urine . Eur . J . Biochem . 134:175-181 . Vyskobil , A , Emminger , S , Tejral , J , Fiala , Z , Etterova , E.andCermanovA . A . (1990 ) Stud yo n kidneyfunctio n i n femal e woeker s expose d t o perchloroethylene . Huma n Exptl . Toxicol . 9:377-380 . Viau . C , Bernard , A , Gueret . F„Maldague . P,Gengoux,P,andLauwen/s , R . (1986 ) Isoparaffini c solvent-induce d nephrotoxicit yi n th e rat . Toxicolog y38 : 227-240 . Wallace , AC , an d Nairn . R. C (1972 ) Rena l tubula rantigen s i n kidne y tumors . Cance r29.977-981 . Warter . H.L . (1983 ) Recen t progres s i n th e pathologica l anatom yo fcancer s o fth e kidney . Progr . Surg . (Basel ) 17:32-57 . Watabe . T , Hiratsuka , A , Ozawa , N , an d Isobe , M . (1981 ) A comparativ e stud yo n th e metabolis m o fd-limonen e an d 4-vinylcyclohex-l-en e b y hepati c microsomes . Xenobiotic a 11:333-344 . Webb , D.R , Ridder , GM , an d Alden . C.L . (1989 ) Acut e an d subchroni c nephrotoxicit

yo fd-limonen e i n Fische r34 4 rats . Fd . Chem . Toxicol . 27 : 639-649 . Webb , D.R , Kanerva , R.L. . Hysell . D.K. , Alden , C.L , an d Lehman McKeeman , L.D . (1990 ) Assessmen t o fth e subchroni c ora l toxicityo f d-limonen e i n dogs . Fd . Chem . Toxicol . 28:669-675 . Wen . OP , Tsai , S.P , McLellan , W.A. , an d Gibson , R.L . (1983 ) Long-ter m mortalit ystud yo

foi l refiner

yworkers : mortalityo fhourlyan d salarie d workers . Am . J . Epidemiol . 118 : 526-542 . Wong , O , an d Raabe . G K . (1989 ) Critica l revie wo fcance repidemiolog y i n petroleu m industr

yemployees , wit h a quantitativ e meta-analysi s b y cance rsite . Am . J . Industr . Med . 15 : 283-310 . Yoon , J.S , Mason , J.M , Valencia , R , Woodruff , E.C , an d Zimmering . S . (1985 ) Chemica l mutagenesi s testin g i n Drosophiia . IV . results ot 4 5 codedcompoundstestedfortheNationalToxicolog y Program . Environ . Mutagen . 7 : 349-367 .

11 7 

# iYu , M.C , Mack , T.M , Hanisch , R , Cicioni , C , an d Henderson . BE . (1986 )

Cigarette smoking , obesity , diureti c use , an d coffee consumptio n a s risk factor s lo rrena l cel l carcinoma . J . Natl . Cance rInst . 77 : 35 1 -356 .

•U S GOVEKNMtN TPUNTIN C OFFIC E mi_ni.ni , i.o«i 5