Carcinogenesi s vol. 8 no . 1 1 pp . 1635-1643 , 198 7 

## Combinatio n experiment s wit h ver y low dose s o

f thre e genotoxi c iV-nitrosamine s wit

h simila r organotropi c carcinogenidty i n rat s

M.R.Berger , D.Schmah

J an d H.Zerfoan 1 Institute o f

Toxicolog y an dChemotherap y an d'institut e o f Experimental Pathology , Germa

n Cance r Researc h Center , ImNeuenheimer Fel d280 , D-690 0 Heidelberg , FR G Th e stud y wa s designe d t o asses s th e syncarclnogen k ac tivity o f very lo w dose s o f

A'-nitrosodiethylamin e (NDEA) , A'-iutrosopyrrolidin e (NPYR ) an d A'-nitrosodiethanolamin e (NDE1A ) i n th e live ro f

180 0 mal e Sprague-Dawk y rats . Th e iV-nitrosamine s wer e administere d throughou tth e rats ' live s individuall y an d i n combinatio n a tthre e logarithmicall y space d dos e level s containe d i n drinkin g water . Th e dos e level s i n th e individual dose—respons e experiments range d from th e lowes tconcentration s o f previou s experiment s (NDEA , 0. 1 mg/kg ; NPYR , 0. 4 mg/kg ; NDE1A , 2. 0 mg/kg ) t o dosage s 1 0 time s lowe ran d comprise d a high , mediu m an d lo w dos e (escalation factor : 3.16) . Th e high dos e o f th e combination con taine d th e thre e nitrosamin e concentration s use d a s th e me diu m dose s o f

th e individua l nitrosamines . Th e mediu m combinatio n dos e resulte d fro m th e combine d administratio n o f th e thre e lowes tdosages , an d th e lo w combinatio n dos e consiste d o f

thre e nitrosamin e dosage s whic h amounte d t o one-thir d o f

th e lo w dosage s respectively . Administratio n o f thes e dosage s wa s associate d with a dose-dependen tincidenc e o f live rcancer : NDE A induce d 45 , 3. 8 an d 2.5% ; NPY R cause d 21.3 , 5 an d 1.3% ; NDE1 A generate d 7.5 , 1. 3 an d 2.5% ; an d th e combination s induce d 16 , 4. 2 an d 1.7 % re spectively . Untreate d control s showe d 0.6 % live rcance rin cidence . Beside s th e liver , th e gastrointestina l tract , th e neurogen k tissue , th e urinar y trac tan d th e hematopoietk an d lymphati c tissu e wer e affecte d b y tumo rincidence s increas e d ove rthato f

controls . Ther e was , however , n o well-define d dos e dependenc y a s with th e live rtumors . Thes e result s in dicate dos e dependenc y o f live rtumo rformatio n eve n atvery lo w exposure level s o f

th e individual agents . Th e carcinogeni c effect s o f th e hepatotropi c A'-nitrosamines summe d u p i n com bination . Th e observe d additivity wa s linear . Dos e levels , whic h alon e woul d presumabl y no thav e bee n carcinogenic , effecte d a significan tcance rris k i n combination . Introductio n Th e numbe

r o f conceivabl e experimenta

l design s fo

r investigatin g combinatio n effect s i

n chemical carcinogenesi s i s almos t infinite

. Thus , fo r tw o o r mor e compound s use di

n combination , differ

ence s ma y exis t wit h regar dt o th e compounds ' physiochemical properties , typ e o f

activit y (loca l o r resorptive) , suppose dmech

anis mof actio n (genotoxi c o r

epigenetic) , potency , mod e o f exposur e (oral , b y inhalation , etc.) , organotropism , an dlas t bu t not least , wit h regar dt o th e respectiv e expose dorganis m(anima l •Abbreviations : NDEA , W-nitrosodiethylamine ; NDE1A , N-nitrosodiethanol

amine ; NPYR , iV-nitrosopyrrolidine ; H/M/LD , high/medium/lo w dose ; H/M / LCD , high/medium/lo w combinatio n dose ; PA S reaction , periodi c acid-Schif f reaction ; PAHs , polycycli c aromati c hydrocarbons

. species , conditio n o f targe t organ , pharmacokineti c character

istic s o f th e individual laborator

y animal , etc.)

. I n vie w o f th e complexit y o f th e numbe

r o f variable s i

t i s importan t

to elaborat e essentia l characteristic s t o fin dan experimenta l desig n tha t i s suite dfo r th e investigatio n o f

combinatio n effect s an

dals o ap plicabl e t o other

individua l compound s andclasse s o f compounds

. Everybody familia

r wit h the subjec t

matte r wil l realize , however , tha t despite meticulou s experimenta l desig n som e uncertaint

y wil l remai n whe n result s obtaine di n on e experimenta l mode l ar e extrapolate dt o asecon dmodel

. Thus , ever y experimental desig n wil l onl y b e a

n approac h t o th e proble mo f combinatio n effect s an donly ver

y clear-cu t

result s will giv e generall y applicabl e con clusions

. Th e presen t

stud y wa s designe dt o investigat e th e syncarcino

geni c effec t

o f thre e hepatocarcinogeni c A'-nitrosamine s and , i n particular , th e possibl e summatio n o f carcinogeni c effects o f

very lo w doses

. In previou s relate dexperiment s o f ou r (1,2 ) an do f othe r group s (3,4) , i

t wa s foun dtha t carcinogeni c effects summe d u p whe n chemica

l compound s wit

h identica l organotropis mwer e administered

. Th e previou s experiment s wer e o f amore quali

tativ e an dat

bes t semiquantitativ e nature , wherea s the presen t study wa s base do n strictl y quantitativ e modalities

. A disadvan

tag e o f suc h experiment s i s that

th e suppose deffect s occur onl y at a n advance dag e o f

th e animals , becaus e administratio n o f lo w dose s implie s lon g induction times o f tumors
. Fo r

di e sam e reaso n th e initial numbe r

o f animal s ha dt o b e comparativel y larg e t o ensur e asufficient

number o f animal s a

t a n advance dage , i.e

. whe n tumor s wer e expecte dt o b e seen . Th e thre e A'-nitros o compound s chose n ha dbee n individuall y teste din dose—respons e studies

. Base do n thes e experiment s i t wa s possibl e t o estimat

e dose s which woul dinduc e < 50 % tumor s i n thei r targe t organ whe

n administere dthroughou t the lif e o f th e animal

. Th e thre e nitrosamine s selecte dwer e A'-nitrosodieth

ylamin e (NDEA* ) (5) , A'-nitrosdiethanolamin e (NDE1A ) (6-8 ) andA'-nitrosopyrrolidine (NPYR) (9)

. It i s well know n tha

t result s obtaine din earlie r

dose-respons e studie s unde r different con dition s ca n b e extrapolate dt o othe r experiment s onl y wit

h limi

tations

. It wa s dierefor e decide dt o repeat th e dose-respons e studie s wit

h separat e administratio n o f th e thre e A'-nitros o com pounds

. Th e doses , however , wer e selecte din arang e whic h covere dth e lowes t

dosage s o f

th e previou s experiment s a s wel l a s dosage s thre e an d1 0 time s lower

. Th e result s o f thes e exper iment s the n forme dth e basi s o f th e quantitativ e calculatio n in th e combinatio n experiment

. Generally , many benig n o r malignan t

spontaneou s tumor s occu r in olde r animals

. T o permi t

relevant evaluatio n o f

th e result s obtaine din di e combinatio n experiment , a n especially larg e untreate dcontro l group wa s used

. Sinc e th e previou s experiment s essentiall y showe dhepatotropi c carcinogeni c activit y o f all thre e A'-nitros o compounds , th e combinatio n effect s wer e evaluate d wit h special regardt o hepatocarcinogenesis , i.e

. th e incidenc e o f hepatocellula

r carcinomas ; ye t

th e whol e spectru mo

f tumor s observe di s presente dan ddiscussed

.

© IR L Pres s Limited , Oxford , Englan d 163 5by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromM.R.Berger , D.Schmfih i an dH.Zerban Material s an d method s Chemicals NDEA , NPY Ran dNDE1 A wer e synthesize dan dsupplie db y D r R.Preussman n (Institut e o f

Toxicolog y an dChemotherapy , German Cance r Researc h Center , Heidelberg , FRG)

. Th e identity an dpurity ( > 9 9 % ) o f th e thre e tes t

compound s wa s verifie db y chromatographi c method s usin g detectio n bot h b y therma

l energ y analysi s an dmas s spectrometry
. Animals and

treatment Eightee n hundre dmal e outbre dSpragu e - Dawle y rat s (Charle s River Breeding , Sulzfeld , FRG) , 100 ± 1 0 day s ol da t th e start o f th e experiment , wer e kep t unde r conventiona l condition s (tw o animal s pe r Makrolo n II I cage ; temperatur e 2 3 ± 2°C ; relativ e humidit y 5 5 ± 10% ; illuminate dperio d1 2 h/day)

. Al l animal s receive daspecifi c nitrosamine-poo r diet whic h containe d< 2 p.p.b

. o f /V-nitroso

dimethylamin e an do f

NPY R(132 0 N , ahromin , Lage , FRG)

. Thi s die t wa s of

fere dad libitum durin g th e earl y stage s o f th e experiment. Afte

r 4 month s th e dail y ratio n wa s restricte dt o 2 1 g pe r animal , correspondin g t o 6 5 kcal , i n orde r t o maintai n amea n bod y wt o f 42 0 g . Th e rat s wer e randoml y stratifie dint o 1 3 experimenta l groups , a s show n i n Tabl e I
. Th e different

grou p size s wer e base do n estimate dtumo r incidences

. Th e Nnitros o compound s wer e administere di n drinkin g wate r

accordin g t o th e dosag e schedul e show n i n Tabl e I for amaximu mperio do f

17 0 weeks

. Th e highes t dosage s o f

th e respectiv e singl e A'-nhrosamine s wer e selecte dt o correspon dt o th e lowes t dosag e o f th e previou s experiments

. They ar e terme d'HD ' (high dose)

. Two logarithmicall y space dlower dosage s terme d'MD ' (mediumdose ) an d'LD ' (lo w dose ) wer e chose n t o amoun t t o one-thir dan done-tent h o f th e H D dosage

. Th e highes t

combinatio n dos e (HCD ) containe dth e thre e nitrosamin e concen tration s use da s th e mediu mdose s o f th e individual Af-nitros o compounds

. Th e mediu mcombinatio n dos e (MCD ) resulte dfro mth e combine dadministratio n o f th e thre e L D dosage s an dth e lo w combinatio n dos e (LCD ) consiste do f thre e nitrosamin e dosage s whic h amounte dt o one-thirdo f th e L D dosage s respectively

. Fres h solution s o f

th e necessary concentration s wer e prepare ddail y fiv e times aweek

. Th e concentratio n o f thes e solution s wa s base do n adaily consumptio n o f 7 m l wate r pe r 100 g body wt
. Ove r

th e weeken dal l rat s receive dplai n tap wate r ad libitum, excep t

fo r thos e animal s tha

t hadno t emptie dthei r

carcinoge n solution

. Untreate dcontrol s receive dta p wate r only

. Al l rat s wer e inspecte dtwic e dail y an dweighe donc e amonth

. The y wer e observe dfo r

thei r entir e lifespa

n an dkille donl y whe n moribund

. Dea danimal s wer e carefully dissected

. Al l macroscoptcall y detectabl e lesion s andtumor s were registered. Th e liver, brain , adrena l an dthyroi dgland s an dal l macroscopicall y visibl e abnormalitie s o f

eac h animal wer e fixe di n formali n an

dexamine dhisto

logicall y b y D r

D.Komitowski (Institut e o f Experimenta l Pathology , German Cance r Researc h Center , Heidelberg , FRG)

. Putativ e preneoplasti c foci o f altere dhepatocyte s were determine di n all animal s kille ddurin g th e firs t

3 0 month s o f

th e experiment. Live r

tissu e wa s take n fro m lobe s free of

macroscopically visibl e tumor s andfixedi n Carney' s solution

. Paraffi n section s wer e staine dwit h hematoxyli n andeosin

. Glycoge n wa s demonstrate d i n paralle l section s b y th e periodi c acid-Schiff reactio n (PAS)

. Foc i o f

altere d hepatocyte s wer e classifie daccordin g t o Bannasc h et al. (10)

. Animal s wit

h foci o f clear an dacidophili c o r

mixe dan dbasophib c cell s wer e counted

. Statistical analysis Th e tumo r

rate s wer e analyze dstatisticall y b y comparin g age-adjuste dexpecte d tumo r incidence s a

t differen t site s wit h observe dincidence s (11)

. Difference s betwee n observe dan dage-adjuste dexpecte dtumo r frequencie s wer e compared , usin g th e x 2 test
. Th e reporte d/"-value s ar e two-taile dexcep t fo r thos e fo r which th e us e o f atren dtes t

i s indicated

. Difference s i n surviva l time s betwee n contro l an dA/-nitrosamine-treate dgroup s wer e assessed , usin g th e two-taile dran k su m tes t accordin g t o Wilcoxo n (12)

. Th e mod e o f additivit y i n th e combinatio n group s (linear additivity versus synergisti c o r antagonisti c additivity ) wa s assesse db y analysi s o f

varianc e method s applie dt o th e frequenc y dat a(13)

. Result s Th e essentia l result s ar e presente din Table s I —VI I an dFigure s 1—3

. Untreate dcontro l animal s ha dahig h mea n life-expectanc y exceedin g 100 0 day s (grou p 1 , Tabl e n , Figur e 1 ) du e t o th e intensiv e car e an dth e goo dgenera l conditio n of th e animals

. Malignancie s wer e diagnose din 29 % o f th e contro l animals , whil e th e numbe r

o f animal s bearin g benig n tumor s a t dissectio n wa s 2.5-fol dhighe r

(72%)

. Th e mea

n tumo r rat e in tumor
bearin g animal s wa s on e malignan t o r tw o benig n tumor s (grou p 1 , Tabl e H)

. Thre e animal s (0.6% ) develope d'spontaneous ' Tabl e I
. Experimental desig n o f administerin g W-nitrosodiethylamin e (NDEA) , JV-nitrosopyrrolidine (NPYR ) andW-nitrosodiethanolamin e (NDE1A) t o male S D rat s Grou p No
. o f

Singl e dos e administered/day* b no . Media n tota l dose* b animal s NDE A NPY RNDE1 A Characte r NDE A NPY RNDE1 A o f dosin g 500--- - __ _ 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c 8 0 8 0 8 0 8 0 8 0 8 0 8 0 8 0 8 0 100 24 0 24 0 0. 1 0.03 2 0.0 1

-

-

-

-

-

- 0.03 2 0.0 1 0.003 2 —

-

- 0. 4 0.13 3 0.0 4

_

-

- 0.1 3 0.0 4 0.01 3 —

-

-

-

-

- 2. 0 0.6 3 0.2 0 0.6 3 0. 2 0.06 3 H D M D L D H D M D L D H D M D L D HC D MC D LC D 61. 1

20. 3 6. 5

-

-

-

-

-

- 21. 0 6. 7 2. 1 —

-

- 27 2 85. 2 26. 4

_

-

- 8 5 2 7 8. 7 —

-

-

-

-

- 132 7 420. 8 130. 9 40 9 13 5 42. 2 •mg/k g bod

y wt
. "W-nitrosamine s wer e administere dvi ath e drinkin g wate r 5 x/week

. TID , hig h dose ; MD , mediu mdose ; LD , lo w dose ; HCD , hig h combinatio n dose ; MCD , mediu mcombinatio n dose ; LCD , lo w combinatio n dose

. hepati c tumors , i.e

. on e hepatocellula r adenoma , on e hemangi

om aandone fibrosarcom a(group 1 , Tabl e IV)

. Th e exceptionally lon g mea n life-expectanc y o f

th e untreate drat s account s fo r th e fac t tha t thes e tumor s were diagnosed , becaus e they were detecte d at amea n lifetime o f

120 0 days

. Al l treate danimal s tolerate dth e administratio n o f th e thre e N-nitros o compound s withou t any symptom s of

toxi c damage

. Th e media

n life-expectanc y o f

treate danimal s i

n th e individua

l group s wa s no t

reduce di n compariso n wit h th e contro l (Tabl e II , Figure s 1 an d2 ) excep t fo r group s 2 aan d2 b (hig h an d mediu mdos e o f

NDEA , Tabl e IT) , in whic h th e media n life

expectanc y wa s slightl y bu t significantl y reduce dby 7 7 and5 2 day s respectivel y ( P < 0.003)

. Regula r administratio n o f

0. 1 mg NDEA/k g bod

y wt induce d a n overal l malignanc y incidenc e mor e tha n twic e a s high a s in th e contro l grou p (65% , grou p 2a , Tabl e II)

. Reductio n of this dos e t o one-thirdo r

one-tent h yielde d3 4 an d29 % malignant tumor s respectivel y (group s 2b , 2c , Tabl e II)

. I n contras t t o thi s dose—respons e relationshi p observe dwit h malignant

tumors , a n eve n distributio n o f

benig n tumor s wa s found , no t significantly different fro mtha t o f controls

. A s expected , neoplasti c change s wer e mos t frequently found in th e liver

. Fo r

instance , 45 % o f

th e animal s treate dwit

h NDEA H D (grou p 2a , Tabl e HI) hadlive r tumors
. Th e tw o lower

dos e schedule s (3 3 an d10 % o f NDE A HD ) yielde dtumor s i n onl y 4 an d2.5 % respectivel y (NDE A M D an dLD , group s 2b , 2c , Tabl e HI)

. Th e incidenc e o f hepati c tumor s wa s significantl y increase di

n all thre e NDEA-treate dgroup s ( P < 0.05). Accord

in g t o th e histological examinatio n o f hepati c tumors , adminis

tratio n o f 0. 1 mg/kg NDE A (HD ) induce dmor e hepatocellula r tumor s (30% ) tha

n tumor s o f

mesenchymal origi n (19% ) (grou p 2a , Tabl e IV)

. A different distributio n wa s seen in th e group s wit h lower incidence s o f

hepati c tumors , probabl y becaus e o f

163 6by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromA combinatio n experimen twith thre e genotox k /V-nitrosamines

Tabl e NPYR Grou p no .

1 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c 11

. Media

n survival time , incidence s o f malignant

an dbenig n

, NDE1 A o r

thei r combinatio n Mod e o f treatmen t

Contro l NDE A H D NDE A M D NDE A L D NPY RH D NPY RM D NPY RL D NDE1 A H D NDE1 A M D NDE1 A L D HC D MC D LC D Media n survival time* b (95 % confidenc e limits )

93 1 (910-945 ) 85 4 (816-892) d 879(812-939) c 914(879-1017 ) 95 1 (854-1001 ) 89 7 (875-948 ) 92 6 (907-989 ) 92 8 (897-985 ) 93 4 (908-990 ) 91 6 (889-968 ) 91 4 (875-983 ) 94 4 (908-983 ) 937(903-965 ) tumor s an dtumo r number s per

tumor-bearin g Percentag e of

animal s wit

h tumor s Malignant (no. ) 2 9 (144 ) 6 5 (52 ) 34(27 ) 29(23 ) 44(35 ) 35(28 ) 29(23 ) 35(28 ) 36(29 ) 36(29 ) 48(48 ) 33(78 ) 31(75 ) Benig n

(no. ) 72(362 ) 66(53 ) 68(54 ) 7 3 (58 ) 69(55 ) 7 4 (59 ) 79(63 ) 80(64 ) 75(60 ) 7 6 (61 ) 76(76 ) 75(181 ) 7 4 (178 ) animal i

n mal e S D rat s
No
. o f tumor s pe r rat 0 Malignan t
1.0 8 ± 0.2 9

. 2 ± 0.4 6

. 0 ± 0.7 5

. 1 ± 0.6 3 .0 6 ± 0.2 4 1. 1 ± 0.3 1

. 0 ± 0. 0 L.0 7 ± 0.3 8

. 2 ± 0.4 7 L.0 6 ± 0.2 6 1. 1 ± 0.3 1 1.0 8 ± 0.2 7 1.09 ± 0.2 9 expose dt o NDEA ,

tumor-bearin g Benig n
1. 7 ± 0.7 7 1. 6 ± 0.6 0 1. 6 ± 0.6 9 1. 7 ± 0.8 2 1. 8 ± 0.9 0 1. 7 ± 0.6 9 1. 5 ± 0.7 2 1.5 ± 0.6 2 1. 8 ± 0.9 2 1. 7 ± 0.8 2 1. 6 ± 0.7 9 1. 6 ± 0.7 7 1.5 ± 0.7 1 "Day s fromstart

o f th e experiment

. b Standar dsignificanc e versu s contro l accordin g t o th e metho do f Wilcoxo n (12)

. c Mea n ±SD

. d / " < 0.00 1 versu s control

. C P< 0.00 3 versu s control

.

too- I

7 5

5 0

2 5 in o E 

# I ° c to o Untr«at« d contro l » NDE A 0.03 2 mgAf l

•NDE A 

### B

1/ 1

5 0

25

- * N ?T?. 0 .-4_'!*gAg

. « NPY R 0.13 3 mgAf l •NDEL A 2. 0
« NOEL * 0 .
•NDEL A 0. 2 mgA g 20 0 40 0 60 0 80 0 100 0 120 0 0 20 0 Tim e (days ) 400 60 0 80 0 100 0 120 0
Fig

. 1

. Survival curve s o f mal e S D rat s i

n pe r cent o f initia l grou p siz e followin g peroral administratio n o f (B ) NDEA , (C ) NPY Ran d(D ) NDEL A i n compariso n wit

h untreate dcontrol s (A)

. Soli dsymbol s represen t live r tumors
.

163 7by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromM.R.Berger , D.Schmah l an dH.Zerba n th e smal l number s (group s 2b , 2c , Tabl e IV)

. Th e gastrointestina l trac t ranke dsecon damon g th e organ s affecte db y NDEA : 31 , 9 an d1 1 % o f

al l rat s wer e foun dt o bear tumor s i n th e digestiv e trac t after administratio n o f

th e thre e NDE A regimens respect
ivel y (group s 2a—c , Tabl e HI) , compare dwit h 5 % i n th e contro l group . Th e difference s wer e significan t (P< 0.01)

. Tumor s in th e ora l cavity an d—i n th e H D grou p —i n th e esophagu s accounte dfor

th e increas e i n tumo r

incidenc e i n th e gastrointes

tinal trac t (group s 2a—c , Tabl e V)

. Renal tumo r incidence s wer e significantl y raise di n th e thre e NDE A groups , althoug h th e observe dfrequencies wer e ver y lo w (1.3 , 1. 3 an d2.5 % i n group s 2a , b , c respectively versu s 0.2 % i n th e contro l group : P< 0.03)

. Th e tes t

fo r dose-relate dtren dwa s significant a s wel l (P
= 0.005)

. Al l th e othe r

organ-relate dtumo r

frequencies di dno t differ significantl y fromth e contro l group . Th e overal l percentag e (44% ) o f al l malignan t

tumor s induce d
10 0

c o 5 0
-

25

- N ? E * J3.O3 2 +_NP\ R O.j j + NDF.L A 0.6JjVng/kg ) NDE A 0.0 1 +

NPY R 0.0 4 + NDEL

A 0.2(mg/kg )

•NDE A 0.003 2 + NPY R 0.01 3 + NDEL A 0.063(mflAg )
20 0 40 0 60 0 80 0

Tim e (days ) 100 0 120

0 Fig . 2

. Survival curve s o f mal e S D rat s i

n pe r cent o f initia l grou p siz e followin g combine dperoral administratio n o f NDEA , NPYRandNDEIA

. Soli dsymbol s represen t

live r tumors
. b y NPY RH D wa s lower tha n i n th e correspondin g NDEA grou p but still 1.5-fol

dhigher tha n i n th e contro l grou p (group 3a , Tabl e II)

. Th e tw o lowe r

dos e schedule s o f

NPY R(NPY RM D an d NPY RLD ) induce dtumo r rate s whic h corresponde dt o thos e recorde din th e respectiv e NDE A group s (group s 3b , 3c , Tabl e II)

. Th e tumo r

incidenc e in th e live r

a s th e primar y targe t

organ wa s 2 1 % afte

r administratio n o f

NPY RH D an d5 % afte

r NPYR MD
. Bot h incidence s wer e significantl y different fromtha t o f th e control s ( P : £0.002 , group s 3a , 3b , Tabl e HI)

. N o signifi

cantl y increase dlive r

tumo r incidenc e wa s observe dfollowin g NPY RL D (1% , grou p 3c , Tabl e III)

. Accordin g t o th e histo

logica l classification , th e majorit y o f NPYR-induce dliver

tumor s wer e o f hepatocellula

r origi n (group s 3a-c , Tabl e IV)

. NPY R administratio n di dno t

increas e th e numbe r

o f tumor s in othe r organs , excep t

for th e M D i

n th e gastrointestina l trac t an dth e L D in th e urinar

y tract
. Th e significan

t increas e in gastrointestina l tumor s followin g NPY RM D wa s du e t o asligh t increas e in tumor s o f th e ora l cavit

y an dth e forestomac h (grou p 3b , Tabl e V) , tha t followin g NPY RLD wa s du e t o araise dfrequenc y o f renal tumor s (grou p 3c , Tabl e HI)

. I n th e cas e o f

NDEI A al l thre e dos e regimens produce da1.2

- fol dincreas e i n malignan t tumor s compare dwit

h th e contro l grou p (group s 4a-c , Tabl e H)

. Henc e n o dos e —respons e re lationshi p i

n term s o f th e overal l malignanc y rat e wa s observed

. Administratio n of

NDEIA , however , resulte din significantly dif

ferent organ-specifi c tumo r

incidence s compare dwit h th e un treate dcontrols

. Th e H D o f NDEI A induce d8 % live r tumor s (grou p 4a , Tabl e HI ; P< 0.00005) , th e M D an dL D induce d onl y 1 % (non-significant) an d3 % ( P < 0.04 ) respectively (group s 4b , 4c , Tabl e HI)

. Tumor s o f hepatocellula

r origi n prevaile daccordin g t o th e histologica l examinatio n (group s 4a—c , Tabl e IV)

. Th e increas e i n gastrointestinal tumor s observe dafte r admin

istratio n o f

th e L D o f

NDEIA wa s significant , thoug h onl y nea r th e limi t of significance

. I t

wa s du e t o th e slightl y highe r rat e o f tumor s i n th e oral cavit

y an din th e forestomac h (grou p 4c , Tabl e m . Grou p no .

1 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c Tumo r

distributio n i

n different Mod e o f treatmen t

Contro l NDE A H D NDE A M D NDE A L D NPY RH D NPY RM D NPY RL D NDEI A H D NDEI A M D NDEI A L D HC D MC D LC D targe t organs * Percentag e Liver
0. 6 (3 ) 4 5 <36f 3. 8 (3) c 2. 5 (2) e 21. 3 (17) c 5 (4 f 1.3(1 ) 7. 5 (6) c 1.3(1 ) 2.5(2 ? 1 6 (16) c 4. 2 (10) c 1. 7 (4 / , b

o f animal s (no

. o

f animals ) wit

h Gastrointestinal trac t
5. 2 (26 ) 31. 3 (25) c 8. 8 (7 y 11. 3 (9f 7.5(6 ) 8.8(7) ' 7. 5 (6 ) 7. 5 (6 ) 3. 8 (3 ) 8. 8 (7) c 9 (9 f 7. 9 (19 f 5. 8 (14 ) tumor s o f th e Neurogeni c tisu e
10. 8 (54 ) 5 (4 ) 13.8(11 / 12. 5 (10 ) 12. 5 (10 ) 11.3(9 ) 6. 3 (5 ) 13.8(11 ) 2 0 (16) ' 1 0 (8 ) 1 7 (17) ' 1 0 (24 ) 12. 1 (29 ) Hematopoiet k an d lymphati c system 4. 6 (23 ) 5 (4 ) 5 (4 ) 2. 5 (2 ) 5 (4 ) 6. 3 (5 ) 7. 5 (6 ) 8. 8 (7) f 6. 3 (5 ) 1 0 (Sf 3 (3 ) 4. 2 (10 ) 7. 9 (19) e Urinar y trac t
0. 2 (1 ) 1. 3 (1) ' 1.3(1) ' 2. 5 (2) d 0 (0 ) 1.3(1 ) 2. 5 (2) d 1.3(1 ) 1.3(1 ) 0 (0 ) 3 (3) d 0.8(2 ) 0. 4 (1 ) •Definitio n a s targe t

organ s wa s base do n significant difference s fromgrou p 1 accordin g to footnot e b

. ''Pooled , age-adjuste danalysi s for heterogeneit

y (two-tailed ) between observe dan dexpecte dtumo r incidence s accordin g t o Pet o et al. (11). C P< 0.00 1 versu s control

. d P< 0.0 1 versu s control

. e P< 0.0 5 versu s control

. { P< 0. 1 versu s control

. 163 8by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromA combinatio n experimentwith thre e genotox k /V-nitrosamines

Tabl e V)

. Additionally , th e rat e o f neurogeni c tumor s wa s sig

nificantl y raise dfollowin g th e administratio n o f th e M D o f NDE1 A (P< 0.03 ; grou p 4b , Tabl e IE) , an dth e numbe r o f tumor s o f th e hematopoietic

- lymphati c syste mwa s significantl y increase db y th e L D o f

NDE1 A (P< 0.02 ; grou p 4c , Tabl e HI)

. Combine dadministratio n o f th e MD s o f

NDEA , NPY Ran d NDE1A , i.e

. th e highes t

combinatio n dosage , wa s associate dwit h a1.7-fol dincreas e in malignant

tumor s (grou p 5a , Tabl e E)

. Thi s paramete r wa s stil l slightl y elevate dafter administratio n o f

th e MCD s an dLCD s (1.14

- an d1.07-fol dincreas e ove r

th e control s respectively ; group s 5b , 5c , Tabl e II)

. In th e live r a s th e mai n targe t

organ o f al l thre e nitrosamines , th e tumo r rate s wer e sig

nificantl y raise dt o 16 , 4 an

d2 % for th e HCDs , MCD s an dLCD s respectivel y (group s 5a-c , Tabl e IE)

. Apart fro mon e fibrosarcom aandon e cholangioma , all th e other hepati c tumor s (93% ) wer e o f hepatocellula

r origi n (Tabl e IV)

. Administratio n o f th e HCD s an dth e MCD s resulte din significantl y increase drate s o f gastrointestina l tumor s (P< 0.0 2 fo r both ) du e t

o th e hig h numbe r o f papilloma s o

f th e forestomac h an do f

squamou s cel l carcinoma s o f

th e ora l cavit

y (group s 5a , 5b , Tabl e HI)

. Th e HC D additionall y le dto asignificant increas e i n neurogeni c tumor s an dtumor s o f

the urinary trac t

(grou p 5a , Tabl e HI ; P

Tabl e IV . Classificatio n of primary liver

tumor s accordin g t o histologi c typ e Grou p Mod e of no . treatmen t Animal s wit h hepatocellula

r tumor s Carcinoma s Adenoma s % (no. ) % (no. ) Animal s wit h mesenchymal tumor s Malignant Benig n hemangioendothclioma s hemangioendothelioma s % (no. ) % (no. ) Animal s wit h Cholangioma s Fibrosarcoma s % (no. ) % (DO. )
1 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c Contro l NDE A H D NDE A M D NDE A L D NPYRH D NPYRM D NPYRL D NDE1 A H D NDE1 A M D NDE1AL D HC D MC D LC D 0

26. 3 0 1. 3 1 5 0 1. 3 3. 8 1. 3 0 1 3 2. 1 0. 8 (2D b (0 ) (1 ) (12) (0 ) (1 ) (3 ) (1 ) (0 ) (13 ) (5) c (2 ) 0. 2 3. 8 1. 3 1. 3 6. 3 2. 5 0 1. 3 0 0 3 1. 7 0. 8 (1 ) (3 ) (1 ) (1 ) (5 ) (2 ) (0 ) (1 ) (0 ) (0 ) (3 ) (4 ) Q) — <0.00 1 0.01 2 0.00 5 <0.00 1 0.00 3 0.09 7 <0.00 1 >0. 1 >0. 1 <0.00 1 <0.00 1 0.01 2 0

18

. 2

. 0 0 1

. 0 0 0 0 0 0 0 (0 ) 8(15) b 5(2 ) (0 ) (0 ) 3(1 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) 0. 2 (1 )

0 (0 ) 0 (0 ) 0 (0 ) 0 0 0 2. 5 0 1. 3 0 0 0 (0 ) (0 ) (0 ) (2 ) (0 ) (1 ) (0 ) (0 ) (0 ) 0 (0 ) 0. 2 (1 )

<0.00 1 0.00 2 >0. 1 >0. 1 0.07 6 >0. 1 0.00 7 >0. 1 >0. 1 >0. 1 >0. 1 >0. 1 0

0 0 0 0 0 0 0 1. 3 0 0. 4 0 (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (1 ) (0 ) (if (0 ) 0

0 0 0 1

. 0 0 0 0 0 (0 ) (0 ) (0 ) (0 ) 5(1 ) (0 ) (0 ) (0 ) (0 ) (0 ) 0. 4 (1 ) 0 (0) •Pooled , age-adjuste danalysi s for heterogeneit

y betwee n observe dand expecte dtumo r incidence s accordin g t o Pet o et al. (11)

. Reporte dP-value s are two
tailed

. b Thre e animal s showe dbot h ahepatocellula

r carcinom aan d amalignant hemangioendothelioma

. c On e anima

l showe dbot

h ahepatocellula

r carcinom aan d acholangioma

.
Tab k V . Grou p no .
1 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c Incidenc e and organ

Mod e of treatmen t

Contro l NDE A H D NDEA M D NDEA L D NPYRH D NPYRM D NPYRL D NDE1 A H D NDH A M D NDH A L D HC D MC D LC D distributio n o f gastrointestinal Percentag e o f

animal s (no

. Oral cavity * Malignant 1 (5 ) 3.8(3 ) 6.3(5 ) 2. 5 a ) 1.3(1 ) 3. 8 (3 ) 2. 5 a ) 2. 5 a ) 0 (0 ) 3. 8 (3 ) 1 (1 ) 2. 1 (5 ) 1.7(4 ) Benig n

0 0 0 1. ; 0 0 0 0 0 0 1 0 0 (0 ) (0 ) (0 ) 1(1 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (1 ) (0 ) (0 ) trac t tumor s
o f animals ) wit

h Esophagus b Malignant 0. 2 5 0 0 0 0 0 0 0 0 0 0. 4 0 (1 ) (4 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (1 ) (0 ) tumor s o f

th e Benig n 0 21. : 0 0 0 0 0 0 0 0 0 0 0 (0 ) »(17 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) (0 ) Stomach 0

Malignan t 1.8(9 ) 3. 8 (3 ) 1.3(1 ) 5 (4 ) 2. 5 a ) 0 (0) 1.3(1 ) 1. 3 (1 ) 1.3(1 ) 1.3(1 ) 

### 2 a ) 1.7(4 ) 1.3(3 ) Benig

n 1.8(9 ) 0 (0 ) 0 (0) 2. 5 a ) 0 (0 ) 3. 8 (3 ) 2. 5 a ) 2. 5 (2 ) 1.3(1 ) 2. 5 a ) 4 (4 ) 0. 8 a ) 2. 5 (6 ) Intestine' '

Malignan t 0. 8 (4 ) 0 (0 ) 2. 5 a ) 0 (0 ) 2. 5 (2 ) 1.3(1 ) 2. 5 a ) 1.3(1 ) 1.3(1 ) 1-3(1 ) 1 (1 ) 1.3(3 ) 0. 8 a ) Benig n

0. 2 0 0 0 1. 3 1. 3 0 0 0 0 0 0. 8 0 (1 ) (0 ) (0 ) (0 ) (1 ) (1 ) (0 ) (0 ) (0 ) (0 ) (0 ) 

## a ) (0 ) No
. o f histologi c diagnose s (mai n grou p no. ) •Squamou s cel l carcinoma : 4(1) , 1(1), 4(3) , 2(4) , 7(5) ; fibrosarcoma : 1(1) , ia) , 3(4) , 1(5) ; adenocarcinoma : 2Q), 2(3) , 2(5) ; papilloma : ia) , 1(5)

. b Squamou s cel l carcinoma : 1(1) , 4(2) , 1(5) ; papilloma ; 17a)

. c Squamou s cel l carcinoma : 7(1) , 6^2) , 2(3) , 1(4) ; fibrosarcoma : 1(1) , ia)

. 1(3) , 1(4) ; adenocarcinoma : 1(1) , ia) , 1(4) ; papilloma : 9{1) , 2a) , 5(3) , 5(4)

. d Squamou s cel l carcinoma : 1(1) ; fibrosarcoma : 2(1) , 2a) , 1(3) , 1(4) ; adenocarcinoma : 1(1) , 3(3) , 2(4) ; hemangioma : 1(1) , 1(3) ; polypus : 2(3) , 1(5)

. 163 9by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromM.R.Berger , D.Schraah l an dH.Zerba n

Tabl e Via . Tumo r

distributio n i n non-targe t organs * Grou p no . Mod e o f treatmen t Contro l Percentag e o f animal s (no

. o f

animals ) wit

h tumor s o f

th e Mammary glan dPancra s Adrenal glan dPituitar y glan dThyroi dglan dReticulo/histiocyti c syste mIntegumenta l syste m 8. 4 (42 ) 13. 4 (67 ) 53. 6 (268 ) 17. 4 (87 ) 23.8(119 ) 5 (25 ) 5. 2 (26 ) 2 a 2 b 2 c 3 a 3 b 3 c 3 a 4 b 4 c 5 a 5 b 5 c NDE A NDE A NDE A NPY R NPY R NPY R NDE1 A NDE1 A NDE1 A HC D MC D LC D H D M D L D H D M D L D H D M D L D 3. 8 6. 3 8. 8 6. 3 13. 8 8. 8 8. 8 8. 8 13. 8 9 11. 3 5. 4 (3 ) (5 ) (7 ) (5 ) (11 ) (7 ) (7 ) (7 ) (11 ) (9 ) (27 ) (13 ) 3. 8 (3 )

11.3(9 ) 6.3(5 ) 1 5 (12 ) 8.8(7 ) 13. 8 (11 ) 11.3(9 ) 1 5 (12 ) 12. 5 (10 ) 1 1 (11 ) 13. 3 (32 ) 8. 8 (21 ) 48. 8

4 5 52. 5 5 0 58. 8 57. 5 57. 5 57. 5 51. 3 5 3 48. 3 5 0 (39 ) (36 ) (42 ) (40 ) (47 ) (46 ) (46 ) (46 ) (41 ) (53 ) (116 ) (120 ) 8. 8

18. 8 18. 8 1 5 16. 3 16. 3 13. 8 18. 8 21. 3 1 9 22. 1 17. 5 (7 ) (15 ) (15 ) (12 ) (13 ) (13 ) (11 ) (15 ) (17 ) (19 ) (53 ) (42 ) 1 5

8. 8 17. 5 21. 3 16. 3 16. 3 18. 8 31. 3 27. 5 1 5 19. 2 25. 4 (12) (15 ) (14 ) (7 ) (13 ) (13 ) (15 ) (25 ) (22) (15 ) (46 ) (61 ) 2. 5

1. 3 3. 8 1. 3 1. 3 1. 3 6. 3 1. 3 5 5 3. 8 2. 1 (2 ) (1 ) (3 ) (1 ) (1 ) (1 ) (5 ) (1 ) (4 ) (5 ) (9 ) (5 ) 1. 3 1. 3 (1 ) (11 ) 2.6(2 ) 5 6. 3 2. 6 1. 3 5 5 3 4. 2 2. 5 (4 ) (5 ) (2 ) (1 ) (4 ) (4 ) (3 ) (10 ) (6 ) "Definitio n a s non-targe t orga n wa s base do n non-significan t difference s fromgrou p 1 accordin g to apooled , observe dan dexpecte dtumo r incidence s a s describe db y Pet o a al. (11)

. Tabl e VIb . Grou p no . , Tumo r

distributio n Mod e o f treatmen t i

n non-targe t

organs " Percentag e o f

animal s (no

. Skeleto n Hear t of animals ) wit

h tumor s o f

th e External auditor

y cana l Peritonea

l cavit y age-adjuste danalysi s

Teste s for

heterogeneit

y betwee n

Lung s Othe r

organ s Contro l 1.6(8 ) 0. 8 (4 ) 0. 8 (4 ) 0.4(2 ) 0. 8 (4 ) 0.4(2 ) 0. 2 (1 ) 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c NDE A H D

NDE A M D NDE A L D NPY RH D NPY RM D NPY RL D NDE1 A H D NDE1 A M D NDH A L D HC D MC D LC D 1. 3

0 1. 3 1. 3 1. 3 0 0 0 0 3 1. 3 1. 3 (1 ) (0 ) (1 ) (1 ) (1 ) (0 ) (0 ) (0 ) (0 ) (3 ) (3 ) (3 ) 1.3(1 )

0 (0 ) 1.3(1 ) 0 (0 ) 1.3(1 ) 0 (0 ) 1.3(1 ) 1.3(1 ) 1.3(1 ) 1 (1 ) 0.4(1 ) 1.3(3 ) 0 (0 )

1.3(1 ) 0 1

. 1

. 0 0 0 0 1 0

. 1

. (0 ) 3(1 ) 3(1 ) (0 ) (0 ) (0 ) (0 ) (1 ) 8(1 ) .3(3 ) 1.3(1 ) 0 (0 ) 0 (0 ) 1.3(1 ) 0 (0 ) 0 (0 ) 0 (0 ) 1-3(1 ) 0 (0 ) 2 (2 ) 0.8(2 ) 1.3(3 ) 0

0 2

. 0 0 0 (0 ) (0 ) 5(2 ) (0 ) (0 ) (0 ) 1.3(1 ) 1

. 0 0 0 0

. 3(1 ) (0 ) (0 ) (0 ) .8(2 ) 0

0 1. 3 0 0 0 1. 3 0 0 0 0. 4 0. 8 (0 ) (0 ) (1 ) (0 ) (0 ) (0 ) (1 ) (0 ) (0 ) (0 ) (1 ) (2 ) 13(1 ) 0 (0 ) 1.3(1 ) 1.3(1 ) 0 (0 ) 2.5(2 ) 1.3(1 ) 0 (0 ) 0 (0 ) 0 (0 ) 0.8(2 ) 0.8(2 ) •Definitio n a s non-targe t orga n wa s base do n non-significan t difference s fro mgrou p 1 accordin g t o apooled , age-adjuste danalysi s fo r heterogeneit

y betwee n observe dan dexpecte dtumo r incidence s a s describe db y Pet o et al. (11)

. < 0.0 3 an dP< 0.00 2 respectively ) an dth e LC D cause da significant increas e i

n tumor s o f

th e hematopoieti c an

dlymphati c syste m(P<0.04)

. Th e dos e —respons e curve s o f al l malignan t

liver tumor s fol

lowin g th e administratio n o f NDEA , NDE1A , NPY Ran dthei r combination s ar e show n Figur e 3

. Interestingly , ales s steep increas e o f

malignancie s wa s foun din th e lowe r dos e range . T o estimat e th e mod e o f additivity , i.e

. apossibl e deviatio n fro msimpl e additivity in a n antagonisti c o r

synergisti c way , th e sum s o f th e tumo r

incidence s followin g th e MD s an dLD s o f th e individua l Af-nitrosamine s wer e compare dwit h th e tumo r incidenc e followin g th e simultaneou s administratio n o f thes e dosages , i.e

. th e HC D an dMC D respectively

. A differenc e o f 6 % wa s foun dbetwee n th e su mo f live r tumo r incidence s ob

serve dafte r

th e mediu mdose s (10% , group s 2 b + 3 b + 4b , Tabl e III ) an dth e liver

tumo r incidenc e followin g the combinatio n HC D (16% , grou p 5a , Tabl e HI)

. Thi s difference , however , wa s no t significant

. Whe n th e su mo f live r tumo r incidence s observe d afte r th e MD s i s correcte dfo r th e backgroun do f

normall y oc currin g live

r tumor s b y subtractin g twic e th e incidenc e o f th e control , i.e

. grou p 5 ai s equate dt o group s 2 b + 3 b + 4 b - ( 2 x grou p 1) , th e differenc e i s mor e accentuate d(7. 2 instea d o f 6% ) bu t still insignificant

. Only whe n comparin g th e respectiv e incidence s of

hepatocellula

r tumor s ( 5 versu s 16% , group s 2 b + 3 b + 4 b versu s grou p 5a , Tabl e IV ) wa s significanc e eviden t i n th e x 2 tes t

( P < 0.02)

. Th e importanc e of thi s finding , whic h woul dindicate overadditiv e synergis min carcinogenesis , i s diminishe db y th e fac t

that th e incidenc e o f

tumor s o f

hepato

cellular origi n wa s merel y additiv e whe n th e sumo f tumo r rate s induce dby th e LD s o f

th e individua

l nitrosamine s (3.7% , group s 2c + 3 c + 4c , Tabl e IV ) wa s compare dwit h th e tumo r

rat e induce dby th e combinatio n MC D (3.8% , grou p 5b , Tabl e IV)

. Wit h regardt o al l live r

tumors , th e rati o wa s 6.3:4.2 % (group s 2c + 3 c + 4 c versu s grou p 5b , Tabl e HI ) o r 5:4.2 % whe n correcte da s describe dabov e an dthu s reversed. Compariso n o f bot h dos e level s suggest s alinea r additivit y o f /V-nitrosamine

164 0by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromA combinatio n experimen twit h thre e genotox k /V-nitrosamines

Tabl e VII . Distributio n of foci o f altere dhepatocyte s i

n relatio n t o tim e Grou p DO .

1 2 a 2 b 2 c 3 a 3 b 3 c 4 a 4 b 4 c 5 a 5 b 5 c Mod e o f

treatmen t

Contro l NDE A H D NDE A H D NDE A L D NPY RH D NPY RM D NPY RL D NDE1 A H D NDE1 A M D NDE1 A L D HC D MC D LC D % sampl e siz e 

# w

2 2 (108 ) 50(40 ) 33(26 ) 25(20 ) 25(20 ) 26(21 ) 25(20 ) 39(31 ) 2 4 (19 ) 21(17 ) 3 1 (31 ) 28(66 ) 27(64 ) 12-2 4 month s

No
. o f animal s withi n perio d
5 6 1 2 1 3 1 2 1 4 1 0 6 1 7 6 6 1 6 2 3 2 7 % animal s wit h clear/acidophili c cel l foci b 1 1 4 2 3 1 1 7 5 7 4 0 1 7 2 9 0 0 4 4 3 0 3 7 % animal s wit h mixcd/basophili c cel l foci " 0 3 3 0 8 7 0 0 0 0 0 0 0 0 25-3 0 month s

No
. o f animal s withi n perio d
5 2 2 8 1 3 8 6 1 1 1 4 1 4 1 3 1 1 1 5 4 3 3 7 % animal s wit h clear/acidophili c cel l foci " 3 7 7 1 3 1 3 8 6 7 4 6 5 0 6 4 6 2 4 6 8 0 6 1 5 7 % animal s wit h mixed/basophili c cel l focib 8 3 2 8 1 3 5 0 9 0 7 7 1 8 7 1 4 1 6 'Animal s (pe r

cen t o f tota l grou p size ) which wer e examine dfor foci o f altere dhepatocytes

. b Percentag e of

animal s of

investigate dsample

. induce dsyncarcinogenicity

. In afurther approac h th e statistical mode l (A ) % incidenc e = a + b\X\ + b^x2 + ^3* 3 (x , = dos e o f

NDEA , x2 = dos e of

NPYR , x 3 = dos e o f NDE1A) wa s formall y fitte dt o th e frequenc y dat aobtaine di n th e respectiv e groups . It s goodnes s o f fi t wa s compare dwit

h tha t o f thi s mode l plu s a

n interactio n termo f

all thre e carcinogen s whic h account s for

non-additiv e influences : (B ) % incidenc e = a + b^x x + £2* 2 + ^3* 3 + *i,2,3*1*2* 3 Th e goodnes s o f

fi t followin g mode l A wa s 0.92 7 (if statistic ) an dtha t followin g mode l B 0.946

. Th e smal l increas e foun d indicate s tha

t th e frequenc y dat aobserve din the combinatio n ca n mostl y b e explaine db y th e assumptio n o f linea r additivity

. Table s Vi aan dVI b presen t th e occurrenc e o f

al l othe r tumo r types , thei r

orga n relatio n an dincidence

. Non e o f thes e tumor s wa s treatmen t

relate ds o tha

t the y ca n b e classifie da s 'spon

taneous'

. Th e incidenc e o f

putativ e preneoplasti c foci o f altere dhepato

cyte s i s summarize dfo

r al l experimental group s an dcontrol s i n Tabl e VH
. Wit

h afe w exceptions , th e numbe r o f animal s wit

h foci o f clea r an dacidophili c glycogen storag e cell s appearin g earl y durin g hepatocarcinogenesi s showe dadose-dependenc e whic h wa s simila r t o tha t o f hepati c adenoma s an

dcarcinoma s (se e Tabl e IV ) developin g later

. Th e highe r th e singl e dose s o f

th e carcino

gen s administere dth e more foci of altere dhepatocyte s emerged

. Thi s hold s tru e for

bot h th e administratio n o f

th e different

nitros

amine s give n alon e an dth e combine dadministratio n o f thes e compounds

. Anothe r

interestin g resul t

i s tha t th e numbe r

o f animal s wit

h clear an dacidophili c cel l foci increase dwit h tim e an dtotal dos e o f

th e carcinogen(s ) applied

. Th e mixe dan dbaso

phili c foci whic h ar e considere dlate r phenotypi c change s i n hepatocarcinogenesi s wer e onl y rarel y see n i n th e secon dyear o f treatmen t

bu t ar e foundin nearl y all experimenta l group s in th e thir dyear . A well-define ddose-dependenc e o f thes e foca

l cel l population s i s no t

evident

. It

shoul dals o b e mentione dthat 70
-

0.0 1 Dos e • respons e relationship s fo ral l malignan t

tumor s o f th e live rfollowin g administratio n o f NDEA , NPYR , NDEL A o rthei rcombinatio n

NDEA

NDEL A

0. 1 1 1 0 Singl e dos e o f N • nitrosamine s (m g / kg ) Fig . 3

. Log-probi t

scal e o n th e dose-dependen t incidenc e o f

al l malignant tumor s o f th e liver followin g administratio n o f NDEA , NPYR , NDEL

A o r thei r combination

. th e number

of treate danimal s bearin g foci o f altere dhepatocyte s i s no t increase dove r

th e backgroun do f

th e control s in som e experimental groups . Thi s i s no t surprising , however , sinc e al l qualitativ e dat ao f

individua l animal s wer e collected , whic h d o no t reflect th e numbe r

andsiz e o f

foc i in thes e animals

. Discussio n Th e presen t

study give s ahighl y accurat e accoun t o f th e dose

respons e relationship s o f

very lo w dose s o f

NDEA , NDEL

A an d NPYR
. Th e mai n targe t

organ s o f

thes e nitrosamine s (NDEA : liver , upper

gastrointestinal trac t

an dkidneys ; NPYR : liver ; NDELA : live r

an dnasa l cavity ; for

review se e 14,15 ) coul db e confirme di

n thi s stud y excep t whe n significantly increase dtumo r 164 1by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromM.R.Berger , D.SchmSh l an d H.Zerfoa

n incidence s wer e foun dat highe r dosage s only , whic h wer e no t include di n ou r

experiment

. Fo r

example , nasal cavit

y tumor s followin g NDE1 A wer e no t observe dbecaus e the y hav e bee n describe dt o occu r

followin g dosage s exceedin g 6 mg/kg onl y (8)

. I t wa s demonstrate di n rat s tha t th e hepatotropi c carcinogen

icit y o f thes e genotoxi c N-nitrosamine s i s additiv e when they ar e administere di n combination

. Thi s wa s als o tru e for nitrosamin e dose s whic h alon e wer e no t (NDE1A , NPYR ) o r

presumabl y (NDEA ) no t

carcinogeni c withi n th e lifespa n o f animal s unde r th e bioassay condition s applie d(LCD , grou p 5c , Table s i n an d TV). Thu s thi s experimen t confirm s an dextend s previou s studie s o n th e syncarcinogenicity o f thes e agent s (1,2)

. Th e importanc e o f

thes e finding s become s mor e eviden t whe n th e dos e rang e use di n thi s study i s compare dwit h nitrosamin e amount s resultin g fro mcertai n life-styl e habits

. Accordin g t o Hoffmann et

al. (16 ; for

review se e 15) , fo

r instance , human s smokin g 2 0 cigarettes/da y inhal e 1 -9 0 /tg NDEA , 30-33 0 /x g NPY Ran d0.3-0. 4 /tg NDElA/m 2 o f bod y surface

. Excep t

fo r NDE1A , thes e amount s correspon dwel l t o th e dosage s use dpe r m 2 o f body surfac e in th e ra t experimen t

(1

—4 2 /tg NDEA , 6-16 8 fig NPY Ran d27-84 0 fig NDE1A) , whic h wer e found t o b e carcinogeni c i n combination

. Th e assessmen t

o f th e carcinogeni c hazar dinheren t i n very lo w dose s o f

/V-nitrosamine s wa s mad e possibl e b y th e lon g sur vival o f al l animal s an db y th e lo w incidenc e o f 'spontaneous ' liver tumor s i n contro l rats
. Sinc e n o reduction o f life-expectanc y wa s foun di n treate dgroups , excep t followin g th e tw o hig h dos

age s o f NDE A (group s 2 aan d2b) , onl y th e cause s of deat h wer e change db y th e treatment-relate dincrease s i n live r cance r inci

dence

. T o achiev e asufficien t surviva l time , mal e rat s wer e use d t o exclud e reduce dlife-expectanc y in th e animal populatio n du e t o tumo r death s foun dmor e frequentl y i n femal e rats , e.g . mam mary glan dneoplasias

. Syncarcinogenesi s i s likel y t o b e affected irrespectiv e o f

th e se x o f th e animals

. Th e observe dlow incidenc e o f 'spontaneous ' live r

tumor s (0.6% ) permitte dasmaller number o f animal s t o b e use d(17 ) an dye t allowe ddiscriminatio n o f smal l increase s in live r

tumo r incidenc e du e t o th e treatment
. Con

sequently , suc h lo w concentration s a s 0.1 4 p.p.m . NDEA , 1. 9 p.p.m . NPY Ro r

2. 9 p.p.m . NDE1A , whic h wer e ingeste dvi a th e drinkin g water , wer e significantl y carcinogeni c i n Spragu e — Dawle y rat s at

sufficientl y lon g exposure . Othe r author s reporte da6—8 % spontaneou s live r tumo r inci

denc e i n Co l wort

h rat s (18 ) an d5-20 % in Fische r F-34 4 rat s (19,20)

. I t

wa s thus considerabl y mor e difficult in thes e exper iment s t o identif

y carcinogeni c effect s o f very lo w dose s o f nitrosamines

. Unde r norma l environmenta l conditions , human s —unlik e laboratory animal s —ar e expose dconcomitantl y o r sequentiall y t o severa l carcinogens

. Syncarcinogenesi s i s therefor e likel y t o b e a n importan t

problem

. W e trie dt o approac h thi s aspec t b y simultaneously administerin g genotoxi c compound s wit h identica l organotropism

. Th e characteristi c organotropis mo f the individua

l compound s wa s no t

change din th e chose n combinations , an d th e cytotropis mwithi n th e live r wa s almos t

limite dto live r

cell s only

. Beside s th e liver , othe r organ s reveale dasignificant in

creas e i n tumo r

incidenc e onl y whe n at

leas t tw o of th e thre e compound s administere di n combinatio n effecte datreatment
relatedincreas e i

n tumo r incidence s (gastrointestinal tract , neuro geni c tissue , urinar y tract ) ove r indivual administration

. In lin e wit h thi s observatio n asignifican t dose—respons e relationship wa s observe donl y fo r

live r tumors ; thi s contrast s wit h al l sig

nificantl y increase dincidence s o f non-live r tumor s whic h wer e foun dfollowin g th e combination s o f NDEA , NPY Ran

dNDE1A

. Th e decreas e fromth e high t o th e mediu mdose s an dfro m th e mediu mt o th e lo w dose s o f th e individual compound s an d o f th e combination s indicate s that th e dos e —respons e curve s clearl y ar e les s steep i

n th e lowe r

dos e range , i.e

. mor e tumor s wer e forme din th e low-dos e rang e than th e stee p decreas e fro m th e hig h t o th e mediu mdose s woul dhav e suggeste d(Figur e 3)

. Th e resultin g graph s sho w ahocke y stic k profil e whic h wa s describe dt o b e indicativ e o f repair (21)

. Repai r

phenomen aar e suppose dt o occur

followin g lo w dosage s o f certai n nitrosamine s suc h a s N-nitrosodimethylamin e an dNDE A (22,23)

. Biochemi

call y les s C^-ethylguanin e ca n b e foundin th e live r

o f NDEA

treate drat s whe

n the animal s are pretreate dwit h NDEA fo

r 5 -1 0 week s a s compare dwit

h unpretreate dcontrol s (24,25)

. Th e C^-ethylguanin e adduc t

i s considere dt o b e promutageni c an d appear s t o b e removedby a n alky l transferase

. Th e percentag e o f removedethylguanine , however , i s lowe r whe n highe r

dose s o f th e carcinoge n are administered

. Thi s saturatio n typ e behavio r o f th e enzym e i s claime dt o b e responsibl e for th e observe d hocke y stic k

shap e o f th e curv e (21)

. O n th e othe r hand , th e observe dprofil e dispute s th e efficienc y o f th e repai r

syste ma t ver y lo w dosage s sinc e liver

tumo r formatio n wa s stil l detectabl e at th e lowes t

dosage s used

. Unlike th e dose-respons e curve s establishe dfo r live r tumors , th e incidenc e o f

esophageal tumor s induce db y NDE A fell fro m 26 % followin g administratio n o f 0. 1 mg/k g (NDE A HD ) t o no t detectabl e level s followin g 0.03 2 mg/k g (NDE A MD ) an d0.0 1 mg/k g (NDEA LD) , i.e

. th e dose—respons e curv e o f esophagea

l tumor s wa s muc h steeper
. Th e reason s fo r thi s discrepanc y betwee n live r

an desophageal tumor s induce db y NDE A remain unclear

. Th e resul t

is , however , in accordanc e wit h ou r previou s experiment s o n NDEA-cause dcarcinogeni c effect s (5) , whe n esophagea l tumor s occurre dat

bu t no t belo w dosage s correspond

in g t o 0. 1 mg/kg (NDEA HD)

. I t differ s fro mresults reported b y Pet o et

al. (18) , wh o foun

dmeasurabl e tumo r

incidence s o f th e esophagu s i

n Colwort h rat s at ados e rang e < 0. 1 mg/kg/day

. I t als o differ s fro

mth e result s reportedb y Lijinsk i et al. (19 ) wh o observe dincrease dfrequencie s o f esophagea l tumor s in Fische r rat s afte r weekly dosage s o f

0.04 5 mg/animal

. Thi s dos e i s in betwee n th e tw o lower dosage s o f

NDE A use din thi s ex periment , whic h di dno t

caus e tumor s o f

th e esophagus

. Th e respons e o f

Colwort h an dFische r

rat s i s obviousl y greate r

tha n tha t o f S D rat s wit

h regardt o th e inductio n o f esophagea

l tumors
. Syncarcinogenesi s b y polycycli c aromati c hydrocarbon s (PAHs ) applie dby skin paintin g t o NMRI mic e wa s demonstrate d previously

. Simila

r linea r additivit y o f th e carcinogeni c effect s o f bot h PAH s (26,27 ) andN-nitros o compound s wa s observed

. Thus , compound s whic h belon g t o th e sam e clas s o f agent s an d hav e acommo n targe t

organ , i.e

. th e ski n o r

liver , sho w linea

r additiv e effects

. Sinc e i

n bot h classe s o f

compound s simila

r o r identica l mechanism s o f

activatio n lea

dt o th e ultimate carcinoge n (28,29) , thi s might

explai n th e observe dlinea r additivity

. When , however , compound s wit h th e sam e targe t

orga n bu t

different metaboli c pathways , suc h a s N-methylnitrosoure aan d7,12-di

methylbenzanthracene , wer e administere dconcomitantl y t o 50

- day-ol dfemal e Sprague—Dawle y rats , th e observe dtumorigen

icit y wa s les s than additiv e (30)

. Th e observation s o n foci o f altere dhepatocyte s precedin g th e developmen t

o f hepati c adenoma s an dcarcinoma s ar e o f con siderabl e interes t

i n vie w o f th e controversiall y discusse duseful

nes s o f thes e lesion s a s endpoint s i n carcinogenicit

y testin g (31)

. Th e result s presente dsuppor t th e concep t

that ther e i s no t

onl y aqualitativ e bu t

als o aquantitativ e precursor—produc trelationshi p betwee n foci o f

altere dhepatocyte s an dhepati c tumor s 164 2by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded fromA combinatio n experimen twit h thre e genotax k JV-nitrosamine s

(32-36)

. In addition , th e observation s ar e in lin e wit h th e pro pose dsequenc e o f

phenotypi c cellular

change s durin g hepato

carcinogenesi s leadin g fromclea r an dacidophili c cel l foci throug h mixe dan dbasophili c cel l foc i t o hepati c adenoma s an dcarci

noma s (10 , M.Enzman n an dP.Bannasch , in preparation)

. Th e fe w exception s t o awell-define ddose-dependenc e o f th e foca l prenoeplasti c lesion s observe dma y b e explaine dby th e fac

t that onl y animal s wit

h foci bu t no t th e numbe r

an dsiz e o f

foci in th e individua

l anima l wer e determine din thi s study

. Thi s point require s adetaile dmorphometri c analysi s base do n amore syste
mati c selectio n o f

live r tissu e t o b e examined

. Sinc e singl e smal l foc i o f altere dhepatocyte s frequentl y als o appea r in untreate d ol danimal s a

n unequivocal prognosi s o f tumo r development

wil l onl y b e possibl e whe n th e volum e densit y o f th e foci i s define d in bot h treate danimal s an duntreate dcontrols

. Wit h thi s reser vatio n in mind , ou r

result s sugges t

tha t th e foci o f

altere dhepato

cyte s ma y wel l b e take n a s endpoint s in carcinogenicity testing

. A revie w b y D.Schmah l o n combinatio n effect s in carcino

genesi s i s i

n preparation

. Futur e experiment s o n syncarcinogeni c effect s o f genotoxi c compound s wit h different

metaboli c acti

vatio n an ddifferent

targe t organ s wil l supplemen t

thi s stud y and elucidate th e state dtheoreticall y possibl e interaction s i n carcino

genesi s in define dbiological models

. Acknowledgement s W e than k D rL.Edler , Institute o f

Epidemiolog y an

d Biometry , Germa

n Cance r Researc h Center , forvaluabl e biostatistica l consultation . Th e skilfu

l technica l hel p o f Mrs . A.Weninger , D.Thei s an d A.Danisma n i s acknowledged . Reference s l.SchmShl.D . andThomas.C . (1962 ) Versuch e zu rKrebserzeugun g a n Ratten be i gleichzeitige rGab e vo n 4-Dimethylamino-azo-benzo l un d Dialhylnitros amin . Naturwisscnschqft, 49 , 521-522 . 2 . Schmaril.D . (1970 ) Experimental investigation s i n syncarcinogenesis . VI . Additio n o f

minimum dose s o f

fou rdifferen tlive rcarcinogen s i n rat s i n live r cance rdevelopment . Z Krebsforsch., 74 , 457—466 . 3 . Montesano.R. , Mohr.U. , Magee.P.N. , HilfrichJ . an d Haas.H . (1974 ) Ad ditiv e effec ti n th e inductio n o f kidne y tumour s i n rat

s treate d wit

h dimethyl nitrosamin e and ethylmethanesulphonate . Br. J. Cancer, 29 , 50-58 . 4 . Tatematsu.M. , Miyata.Y. , Mizutani.M. , Hananouchi.M. , Hirose.M . an d Ito , N . (1977 ) Summatio n effec to f A'-butyl-AK4hydroxybutyl)nitrosamine , N- (4-(5-nhro-2-furyI)-2-thiazolyl)fonnamide , Af-2-fluorenylacetarmde , an d 3,3' - dkhlorobenzidin e o n urinar y bladde rcarcinogenesi s i n rats . Gam, 68 , 193-202 . 5 . Druckrey.H. , Schildbach^A. , Schmfihl.D. , Preussmann.R . an d Ivankovic.S . (1963 ) Quantitativ

e Analys e de rcardnogene n Wirkung vo n DiSthylniUusaiiuii . Arauinu-Forsch., 13 , 841-851 . 6 . Preussmann^ . (1962 ) Noti z Qbe reinig e neu e Dialkybiitrosamine . Chen. Ber., 91 , 1171-1172 . 7 . Druckrey.H. , Preussmarm.R. , Ivankovic.S . an d Schmihl.D . (1967 ) Organo trop e carcinogen e Wirkun g be i 6 5 verschiedene n A'-Nitrosoverbindunge n an BD-Ratten . Z Krebsforsch., 69 , 103-201 . 8 . Preussmann.R. , Habs.M. , Habs.H . an d Schmard.D . (1982 ) Carcinogenicit y o f N-nitrosodiethanolamin e i n rat s a tfiv e differen tdos e levels . CancerRes., 42 , 5167-5171 . 9 . Preussmann.R. , Schmahl.D . and Eisenbrand.G . (1977 ) Carcinogenicity o f A'-rutrospyrrolidine : dose—respons e stud y i n rats . Z Krebsforsch. ,90 , 16 1 - 166 . 10 . Bannasch.P. , Zerban.H . an d Hacker. H J . (1985 ) Foc i o f altered hepatocytes , rat . I n Jones.T.C , Mohr.U . an d Hunt,R.D . (eds) , Monographs on Pathology of Laboratory Animals, Digestive System. Springer , Berlin , pp . 10-30 . ll.Peto.R. , Pike.M.C , Day.N.E. , Gray.R.G. , Lee,P.N. , Parish.S. , PetoJ. , Richards,S . an

d Wahrendorf

J . (1980 ) Guidelines fo rsimple , sensitiv e sig nificanc e tests fo rcarcinogeni c effect s i n long-ter m animal experiments . I n IARC Monographs on the Evaluation of the Carcinogenic Risk of

Chemicals to Humans, Suppl. 2. Long-term and Short-term Screening Assays forCarcinogens, a Critical

Appraisal. IARC , Lyon , pp . 311—426 . 12 . Hollander.M. and Wolfe.D.A . (1973 ) Nonparametric Statistical Methods. Joh n Wiley , Ne w York . 13 . Searle.S.R . (1971 ) LinearModels. Joh n Wiley , Ne w York . 14 . IAR C (1978 ) IARC

Monographs on the Evaluation of the Carcinogenic Risk to Humans. Vol. 17, Some N-nitroso Compounds. IARC , Lyon . 15 . Preussmann.R . an d Eisenbrand.G . (1984 ) N-Nitroso Carcinogens in the Environment, American Chemica l Society Monograp h no . 182 . ACS , Washingto n DC . 16 . Hoffmann,D. , AdamsJ.D. , Brunnemann.K.D . and Hecht.S.S . (1982 ) In Scanlan.P . an

d Tannenbaum,S . (eds) , N-Nitroso-Compounds, AC S Symposiu m Scries no . 174 . ACS , Washingto n DC . 17 . ECETO C Moo . no . 2 (1980 ) Europea n Chemical Industr

y Ecolog y and Toxi colog y Centre , Brussels . 18 . Peto.R. , Gray.R. , Brantom.P . and Grasso.P . (1984 ) Nitrosamin e carcino genesi s i n 512 0 rodents : chroni c administratio n o f sixtee n differen tconcen tration s o f

NDEA , NDMA , NPY Ran d NPI P i n th e wate ro f 444 0 inbre d rats , with paralle l studie s o f NDE A alon e o f

th e effec to f

th e ag e o f

startin g (3 , 6 o r2 0 weeks ) an

d o f specie s (rats , mic e o rhamsters) . I n O'Neill^.K. , vo n Borstd.R.C , MiUer.C.T. , LongJ . an d Bartsch,H . (eds) , N-Nitroso Compounds: Occurrence, Biological Effects and

Relevance to Human Cancer. IARC , Lyon , IAR C Scientifi c Publication s no . 57 , pp . 627-665 . 19 . lijinsky.W. , Reuber.M.D . an d Riggs.C.W . (1981 ) Dos e respons e studie s o f carcinogenesi s i n rat

s b y nitrosodiethylamine . CancerRes., 41 , 4997 - 5003 . 20 . Lijinsky.W . an d Kovatch.R.M . (1985 ) Inductio n o f live rtumor s i n rats b y nitrosodiethanolamin e a

tlos e doses . Carcinogenesis, 6 , 1679-1681 . 21.Hoel,D.G. , Kaplan.N.L . an d Anderson.M.W . (1983 ) Implicatio n o f nonlinea r kinetic s o n risk estimatio n i n carcinogenesis . Science, 219 , 1032-1037 . 22 . Montesano.R. , PeggAE . an d Margison.G.P . (1980 ) Alkylatio n o f DN A an d carcinogenicit y o f

A'-nitros o compounds . / . Toxicol. Environ. Health, 6 , 1001-1008 . 23 . Montesano.R. , Bresil.H. , Planche-Martel,G. , Margison.G.P . an d Pegg,A.E . (1980 ) Effec to f

chroni c treatmen

to f rat s wit h dimethyhiitrosamin e o n th e remova l o f

O^-methylguanin e fro m DNA . CancerRes., 40 , 452-458 . 24 . Margison.G.P. , Curun.NJ. , Snell.K . an d Craig^A.W . (1979 ) Effec to f chroni c MA'-diethylnitrosamin e o n th e excisio n o f

O*-ethylguanin e from ra tlive r DNA . Br. J. Cancer, 40 , 809-813 . 25 . Margison.G.P. , Craig.A.W. , BresU^H. , Curtin.N. , Snell.K . an d Montesano,R . (1979 ) Chroni c administratio n o f A'-nitrosamine s enhance s th e remova l o f (Aalkylguanine s from ra

tlive rDN A in vivo. Br. J. Cancer, 40 , 815-816 . 26 . Schmidt.K.G. , Schmahl.D. , MisfekU . and TimmJ. (1976 ) Experimentell e Untersuchunge n zu rSyncarcinogcnese . VII . Mitteilung : Syncarcinogen e Wirkun g vo n polycyclishe n aromalische n Kohlenwasserstoffe n (PAH ) i m Epicutantes ta

n de rMiusehaut . Z Krebsforsch., 87 , 9 3 -100 . 27 . SchmSl.D. , Schmidt.K.G . an d Habs.M . (1977 ) Syncarcinogeni c actio n o f pofycycli c hydrocarbon s i n automobil e exhaus tga s condensates . I n Mohr.U. , Schmahl.D . and Tomatis.L . (eds) , AirPollution and Cancerin Man. IARC , Lyon , IAR C Scientifi c Publication s no . 16 , pp . 53-59 . 28 . Preussmann.R . and Stewart,B.W . (1984 ) I n Scarie.C.E . (ed.) , ChemicalCarcinogens, Vol

2, AC S Monograp h no . 182 . ACS , Washingto n DC . 29 . Grimmer.G . (1983 ) Environmental Carcinogens: Potycydic Aromatic Hydrocarbons. CR C Press , Boc a Raton , FL . 30 . Berger.M . an

d Kflnstler.K . (1983 ) Experiments o n th e influenc e o f combine d applicatio n o f

A'-methylnitrosoure

a an d 7,12-dimethyIbenzanthracene i n femal e Sprague-Dawle y rats . Arch. Geschwulstforsch., 53 , 423-427 . 31 . Bannasch.P . (1986 ) Preneoplasti c lesion s a s en d point s i n carcinogenicit y testing . I . Hepati c preneoplasia . Carcinogenesis, 7 , 689—695 . 32 . Emmelot.P . and Scherer.E . (1980 ) Th e firs trelevantcel l stag e i n ra tlive r carcinogenesis . A quantitative approach . Biochim. Biophys. Ada, 605 , 24 7 — 304 . 33 . Kunz.W. , Schaude.G. , Schwarz.M . an d Tennekes.H . (1982 ) Quantitativ e aspects o f drug-mediate d tumou

rpromotio n i

n live ran d its toxicologica l impli cations . I n Hecker.E. , Fusenig.N. , Marks.F . and Kunz.W . (eds) , Carcinogenesis —a Comprehensive Survey. Rave n Press, Ne w York , pp . 11 1 -125 . 34 . Moore.M.A. , Mayer.D . and Bannasch.P . (1982 ) Th e dose-dependenc e an d sequentia l appearanc e o f

putativ e preneoplasti c population s induce d i n th e ra tlive rb y sto p experiment s wit h N-nitrosomorpholine . Carcinogenesis, 3 , 1429-1436 . 35 . VesseUnovitch.S.D . an d Mihailovrch.N . (1983 ) Kinetic s o f diethylnitrosamin e hepatocarcinogenesi s i n th e infan tmouse . CancerRes., 43 , 4253—4259 . 36 . Goldsworthy.T. , CampbeU.H.A . and Pitot.H.C . (1984 ) Th e natura l histor y an d dose—respons e characteristics o f enzyme-altere d foc i i

n ra tlive rfollowin g phenobarbita

l an d diethylnitrosamin e administration . Carcinogenesis, 5 , 6 7 — 71 . Received on March 30, 1987; accepted on August

10, 1987

164 3by Michelle Cawley on September 6, 2010carcin.oxfordjournals.orgDownloaded from