Influence of strain and age on the induction of mammary tumours by diethylstilboestrol in C3H mice

Fd Chem. Toxw. Vol 22, No. 11. p p 871 874. 1984

Printed in Great Britain, All rights reserved

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0278-6915.84 $ Y 0 0 + 0 . 0 0 1984 Pergamon Press I.Id

I N F L U E N C E OF S T R A I N A N D A G E ON THE I N D U C T I O N OF M A M M A R Y T U M O U R S BY D I E T H Y L S T I L B O E S T R O L IN C3H MICE D. L. GREENMAN, R. L. KODELL, B. HIGIIMAN, G. J. SCHIEFI-RSTEIN and M. NORVEI,I,* Food and Drug Administration and Pathology Services Project. National Center for Toxicological Research, Jefferson, AR 72079, USA (Received 26 March 1984)

Abstraet--C3H/HeJ and C3H.:HeN female mice were fed diets containing targeted concentrations of 320 or 640ppb diethylstilboestrol (DES) starling at 7 or 11 wk of age and continuing throughout their remaining lifespan. Regardless of the DES concentration there was a faster rate of development and higher final incidence of mammary adenocarcinomas among the C3t t..tleN mice than among the C3H:t teJ mice. In C3H/HeN mice started on DES when 11 wk old, mammary tumours developed more rapidly than when treatment was started at 7 wk of age. This was also true for C3H:HeJ mice given 320 ppb DES but not for those treated with 640ppb DES. Both age at the start of treatment and strain of ('31! mice are important factors to be considered in designing experiments to study the tumorigenic activity of oestrogens such as DES.

INTRODUCTION

In earlier studies at this l a b o r a t o r y two strains of C3H mice, b o t h p u r p o r t e d to have high titres of the murine m a m m a r y t u m o u r virus factor ( M M T V ) , were found to develop m a m m a r y t u m o u r s at very different rates when fed diets c o n t a i n i n g diethylstilboestrol (DES). F o r example, 93"0 of a starting p o p u l a t i o n o f C 3 H / H e N virgin females fed a diet c o n t a i n i n g 6 4 0 p p b DES had developed palpable m a m m a r y a d e n o c a r c i n o m a s after 39 wk of DES exposure ( G r e e n m a n & H i g h m a n , 1982) whereas only 29'~,o of a starting p o p u l a t i o n o f C 3 H , H e J virgin females had developed palpable m a m m a r y aden o c a r c i n o m a s after 3 9 w k of exposure to 1 0 0 0 p p b dietary DES ( H i g h m a n , G r e e n m a n , Norvell et al. 1980). These studies were not carried out concurrently and, in a d d i t i o n to the ditterences in strain and dosage, the ages o f the mice at the start of DES t r e a t m e n t differed somewhat. C 3 H : H e N mice were treated from 4 - 5 wk o f age, whereas C3H.:HeJ mice were treated from 6 wk of age. A l t h o u g h separate studies indicate that C 3 H ; H e N (Heston, Deringer & D u n n , 1956) and C3H.:HeJ ( R i c h a r d s o n , 1973) female mice differ from each o t h e r in the incidence and time to a p p e a r a n c e of s p o n t a n e o u s m a m m a r y tumours, no reports have been found in which these two strains have been evaluated simultaneously u n d e r identical environmental conditions for differences in s p o n t a n e o u s or oestrogen-induced m a m m a r y tumorigenesis. Neither could we find any reports of studies in which the

*Current address: FMC Corporation. Chemical Research Development Center, Princeton, New Jersey, USA. Abbreviations: DES = diethylstilboestrol: DM BA = 7,12-dimethylbenz[a]anthracene; HPLC = high-pressure liquid chromatography: MMTV = murine mammary turnout virus factor; NCTR = National Center for T o y icological Research.

influence of age at the start of oestrogen treatment on the rate of mammar3.' t u m o u r a p p e a r a n c e had been examined in either strain. The current stud3,' was designed to determine the relative i m p o r t a n c e of these two factors, strain and age, in designing protocols for evaluating the carcinogenic potential of oestrogens. EXPERIMENTAL Specific pathogen-free C3H,'HeN mice were produced at the N a t i o n a l Center for Toxicological Research ( N C T R ) ; C 3 H / H e J mice were obtained from Jackson Laboratories (Bar H a r b o r , M E ) and were q u a r a n t i n e d for 2 wk before experimental use. DES, o b t a i n e d from Sigma Chemical C o m p a n y (St Louis, MO), was found to be free of impurities when analysed by gas c h r o m a t o g r a p h y and high-pressure liquid c h r o m a t o g r a p h y (HPLC). The DES comprised virtually 100°~, of the trans isomer. The diet (5010M) was purchased from Ralston Purina C o m p a n y (St Louis, M O ) and was found to contain < 5 p p b (b = 10'7) DES equivalents when tested by a uterine weight assay. The diets were autoclaved and reg r o u n d before mixing with DES in a Patterson Kelly V-blender. A solution of DES in 95% ethanol was t h o r o u g h l y mixed with diet in a ratio of 5 ml:100 g ol" feed to obtain targeted dietary DES c o n c e n t r a t i o n s of 320 and 6 4 0 p p b . Ethanol was then removed by mixing u n d e r vacuum at 82 C. All batches o f feed were analysed tbr DES by electron-capture gas chrom a t o g r a p h y or H P L C (King. Nony & Bowman, 1977). M e a n ( + SD) c o n c e n t r a t i o n s for the tv,'o target c o n c e n t r a t i o n s were 331 _+ 56 and 636 _+ 47 ppb. The formulated diets were stored at room t e m p e r a t u r e in stainless-steel containers for 8 wk or less. Virgin female C 3 H . : H e N - M T V + :Nctr ( C 3 H : H e N ) and C 3 H , H e J mice were continuously fed diets containing the targeted DES c o n c e n t r a t i o n s of 320 or 640 p p b starting at either 7 wk (49 54 days) or I 1 wk 871

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(51 52 days) of age and c o n t i n u i n g t h r o u g h o u t their remaining lifetimes. G r o u p s of 72 and 48 mice of each strain were used at the 320 and 6 4 0 p p b levels. respectively.. ('3H..HeN and C3H H e J mice were kept on different cage racks. Each column on a rack was c o m p o s e d of six cages and was r a n d o m l y assigned m t o w to a given treatment g r o u p so thai mice in till cages in a c o l u m n 'aould receive the same experimental diet and animals for a given treatment group would be equall~ distributed on each shell level. Mice from a given strata were assigned to cages r a n d o m l y in a way that virtually eliminated the housing of litlermates in the same cage. The mice were housed four per cage on h a r d w o o d chip bedding in phtstic "shoe-box" type cages with spun polyester filter tops. The animal room was m a i n t a i n e d at 21.1 23.3 C a n d 40-60<',, relative humidity with fluorescent lighting 1 2 h r day (06.00 18.00 hr). Ventilation provided lit • 15 room air changes..hour. Feed and pasteurized ultraliltered water were provided ad liD. l)aily checks were made for dead or m o r i b u n d animals. At weekly m t c r \ a l s animals were weighed and examined for clinical signs of disease or significant changes m their a p p e a r a n c e and then transferred to clean cages with clean feeders and water bottles. Mice were killed and autopsied when palpable body masses (presumptive mammar.'~ turnouts) attained a 1-cm diameter. During the autopsy of mice ttml were removed from the experiment because they died, became m o r i b u n d or developed l-cm masses, the m a m m a r y glands and palpable s u b c u t a n e o u s masses were excised and fixed for 24 hr in Bouin's sohition. They were then trimmed, washed, processed in an a u t o m a t i c tissue processor on a 4-hr schedule, e m b e d d e d in parattin blocks, sectioned at 5 t~ m and stained with haematoxylin and eosin {Frith. H i g h m a n & K o m i c k a , 1976). The classilication system of l ) u n n (1959) was used to identil} m a m m a r y a d e n o c a r c i n o m a s . Probability distribution curves for time of removal from experiment with palpable, histologically-verified type A. B a n d o r C m a m m a r y a d e n o c a r c i n o m a s , adjusted for removal by' c o m p e t i n g risks, ,acre estimated (Kodell, S h a w & J o h n s o n , 1982) and plotted against time. Tests to determine dose-related effects (Pete, Pike, Day e t a / . 1980) ,,,,'ere performed. All estimators and statistical tests were calculated using N C T R ' s SAS procedure C H R O N I C (Kodell, ltaskin, Shaw & Gaylor, 1983). Strain c o m p a r i s o n s ,,,,'ere one-sided since previous studies at N C T R indicated that C31[..IleN mice might be removed with l u m o u r s earlier than C 3 H . H e J mice. These earlier studies also indicated that mice started on DES at a young age might develop t u m o u r s more quickly than mice started at an older age. Thus, a l t h o u g h the current study' does not substantiate this finding, it was decided a priori that statistical c o m p a r i s o n s of the effect of age at the start of treatment would be one-sided.

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Virtually all of the m a m m a r y a d e n o c a r c i n o m a s were of types o f A or B. In general, type B aden o c a r c i n o m a s appeared earlier and were more pre-

d o m i n a n t than type A a d e n o c a r c m o m a s . In ( ' 3 H . H e N mice the B:A ratio was between 7.9:1 and 10.1:1 whereas m C3H.I-|eJ mice ttlis ratio was 3.3:1. Strain differences m m a m m a r y tumorigene~,is arc illustrated m F i g I. Differences belwecn strains in the rate

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highly significant (P < 0.00005 wittl one-sided test):.it both dietary' c o n c e n t r a t i o n s of DES (321) or 640 p p b l and regardless of the age al ~.hich I)I!S feeding x~as initiated (7 or I I wk). In all cases, nmmmary tumours developed earlier in the ('31{.IIeN mice than the\ did m the ('31.t..tleJ mice. A h h o u g h the time until the first m o u s e

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a given dose group x~as onl,,. 40 70 da,,s earlier in ('31{..fteN than it ,aas m ( ' 3 H . H e J mice, Ihc subsequent rate of development of ll]anllllal~, lumours was much slower in ( ' 3 H HcJ mice. Thu,,. the cslimaled lime It) a 50",, incidence of m a m m a r \ lumours was at least 260 days greater in (31-t He,I than it ~.~.as in ('3H. HeN mice for a given dietarx concentration of DES. There was a dose-related reduction in lime of removal

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ment was started al 7 w k (big. la). but liOt al I I x~k of age IKig. IbL It is clear l'rom t-igs 2 & 3 that mice started o n 1)l-'.S at the earlier age did not develop m a m m a r , . l u m o u l s more rapidly than mice started at the later a g e Indeed, in three out of four comparisons, mice started on DES at II u k of age were remoxed with palpable m a m n l a r ~ t u m o u r s after a shorter time of exposure than were mice s h u t e d on DES ',~hen 7 ~k old. This was true tBr C 3 H . H e N mice al bolh DES concentrations and for ( ' 3 1 1 l t e J mice al 3 2 0 p p b I)I{S.

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taining DES continuously., beginning at (al 7 ~k of age or (bY I I w.k of age: ('3H.HeN mice 640 ppb {~), 320 ppb (At: C31I.HeJ mice - 640 ppb (,"_'.,). 320 ppb (:',L l)a,,s on experiment refer to the time elapsed ,,race ~eaning (21 26 days old). Mammary lumour probabilit} is lhc probabihl.,, of an animal being remo,.ed with a palpable, hisloh)gically verified mammary i l d e n o c a r c i n o m l l { s e e Experimental)

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Fig. 2. Effect of age at the start of DES exposure on mammary tumorigenesis• C3H..HeN femalc mice were continuously cxposed to (a) 320 or (b) 640 ppb DES in the diet starting at either 7 (O) or I1 (Q) wk of agc. The duration of exposure to DES is plotted against the probability of a mouse being removcd from the experiment with a palpable, histologically veritied mammary adenocarcinoma (see Experimcntal).

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Fig. 3. Effect of age at the start of DES exposure on mammary tumorigenesis. C3H.:HcJ female mice were continuously exposed to (a) 320 or (b) 640 ppb DES in the diet, starting at 7 (O) or 11 (Q) wk of age. The duration of exposure to DES is plotted against the probability of a mouse being removed from the experiment with a palpable, histologically' verified mammary adenocarcinoma (see Experimental). DISCUSSION AND CONCLUSIONS It is clear from this study that M M T V - p o s i t i v e C 3 H / H e N female mice develop m a m m a r y aden o c a r c i n o m a s in response to DES at a m u c h faster rate than do C 3 H / H e J fcmale mice. O t h e r isolated studies have given evidence that these two strains have different rates and incidences of m a m m a r y t u m o u r development. Heston, Deringer & D u n n (1956) reported a m a m m a r y t u m o u r incidence o f a b o u t 95-100% in untreated M M T V - p o s i t i v e C3H females with an average t i m e - t o - t u m o u r of a b o u t 8 m o n t h s in breeders and 11 m o n t h s in untreated virgins. On the other h a n d . u n t r e a t e d C3H.'HeJ breed-

ing female mice have been reported to have a b o u t a 60% incidence of m a m m a r y t u m o u r s and an average t i m e - t o - t u m o u r of 11 months, whereas virgins had an a p p r o x i m a t e 30% incidence, and a 2 0 - m o n t h average for t i m e - t o - t u m o u r (Richardson, 1973). No other study could be found in the literature in which these two strains have been simultaneously' evaluated with respect to either s p o n t a n e o u s or induced m a m m a r y tumorigenesis. However, differences in the immunological system have been d e m o n s t r a t e d for these two strains (Tagliabue, Ruco, McCoy' c t a l . 1978), and it is well established that i m m u n e competence can have a m a r k e d effect upon carcinogenesis (Anisimov & Turusov, 1981 ). Age at the start of dosing, in the present experiment, did not have an effect on t i m e - t o - t u m o u r that could account for differences that have been noted between strains in earlier experiments carried out at this l a b o r a t o r y ( G r e e n m a n & H i g h m a n , 1982, Highm a n et al. 1980). F r o m those two studies, one might have predicted that animals started on DES at a s o m e w h a t older age would have developed l u m o u r s much more slowly than those started earlier. This clearly was not true for either strain in the present study. Indeed, for both strains at 320 ppb DES and for C 3 H / H e N mice at 640 p p b DES m a m m a r y Ium o u r s developed after a shorter d u r a t i o n of exposure in mice started on treatment at 11 wk than in those started at 7 wk of age. This observation suggests that between 7 and 11 wk of age both strains of mice arc refractory to DES treatment. A similar period during which SLN mice are apparently resistant to the effect of prolactin on m a m m a r y t u r n o u t development has been reported (Nagasawa. 1983). F u r t h e r m o r e , this finding is reminiscent of observations made on the induction of mammary' a d e n o c a r c i n o m a s in female rats by 7,12-dimethylbenz[a]anthraccne (1)MBA: Janss & Hadaway, 1977: Mcranze, G r u c n s t e m & Shimkin, 1969). In rats sensitivity to t u m o u r induction is highly age dependent, the precise age dependence being strain specific, Ibr example, m studies with D M B A S p r a g u e - D a w l e y rats were more sensitive to t u m o u r induction at 50 than at 80 days of age whereas L o n g - E v a n s rats were insensitive at 50 days but sensitive at 80 days c,f age (Janss &

S74

1.). L. (jREENMAN Cl gIL

l l a d a w a y , 1977). R a t e s o f D N A s y n t h e s i s o r Ill~.inlm a r y g l a n d g r o w t h were f o u n d to be h i g h e r d u r i n g s e n s i t i v e p e r i o d s t h a n d u r i n g i n s e n s i t i x e p e r i o d s in h o t h rat s t r a i n s a n d in SI.N mice ( J a n s s & H a d a w a y , 1977- N a g a s a w a . 1983). W h e t h e r s i m i l a r d i f f e r e n c e s in t h e rate o f g r o w t h o r D N A s y n t h e s i s a r e a s s o c i a t e d with d i l l e r e n c c s in s e n s i t i v i t y to D E S in C 3 H mice n e e d s to he e x a m i n e d . Since p r o l a c t i n is knm~.n to bc o f p r i m a r y i m p o r t a n c e m m a m m a r y t u m o r i g e n e s i s in t h e m o u s e I W e l s c h & INaga.saw;.l. 1977i a n d D E S h a s h c c n shov, n to i n c r e a s e t h e level o f c i r c u h t t i n g p r o kiclin ( S i n h a . Sclby, l.ewis & V a n d e r l a a n , 1972) t h e r e f r a c t o r y p e r i o d n o t e d in t h e p r e s e n t s t u d ~ c o u l d be e x p k i i n e d either by a lkiiltire oF D E S to i n c r e a s e c i r c u l a t i n g p r o l a c t i n o r a f a i l u r e o f the nl;.imn'lary g l a n d It) r e s p o n d to p r o l a c t i n d u r i n g lhe r e f r a c t o r y period. P o s s i b l e a g e d i f f e r e n c e s in M M T V titre c o u l d also be a f a c t o r t h a t m e r i t s i n v e s t i g a t i o n . In c o n c l u s i o n , age tit the s l a r t o f t r e a t m e n t ~ i t h D E S i n l l u e n c e s n'lan'lnlar 5 t u m o u r d e v e l o p m e n t in C 3 1 t . . t t e a a n d ( ' ] H HeJ f0nlale mice. H o w e v e r , the s t r a i n o f m o u s e is a m u c h m o r e J m p o r l a n l v a r i a b l e in d e t e r m i n i n g the rale a n d i n c i d e n c e o r m a m m a r y i u m o u r de~,elopment.

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Anisn'nm V. N. & T u r u s m V. S. (19~;I). Modil~.ing ctt'ect of ageing on chemical carcinogenesis. A review...llech. .4ge. I)er. 15, 399. Durra T. B. 11959). Morphology of m a m m a o tumors m mice. In The Phwiopathoh~g.v ol Cancer. Edited b~ F. ttomberger. 2nd I".d.p. 38. IIoebcr Ilarper. Nev, Y'ork. Frith ('. H.. Highman B. & Konvicka A. (1976). Ad,,ances m automation li~r experimental pathology, l,ah..4him. S~t. 2@ 171. Greenman D. L. & Highman B. 11982). N ( I R l~,chnical Report /or E.,;periment 07g: Final Report: Carcinogenicl'Atrogemc Bioav~m with ('311 Mice. National ('enter for Tt~xicological Research. Jefferson. AR. Heston W. t!.. I)eringer M. K. & Dunn T. B. (1956). Further studie~, on the relationship between the genotype and the mammary tumor agent in mice..I, mils. Cancer ln~l. 16, 1309.

Highman B,. Greenman I). I... Nor~ell M .I.. l a n n m J. & Shellenberger T I{. (198(I). Neoplastic and prcncoplastic lesions reduced m female ('3tt mice b', dwt~, ,,.'t)nt~linlng dieth.,,.lstilbestrol or 17fl'-eMradiol. J. cnrir Path lbxtcol. 4 (5 & 6L 81. janss D. It. & Ilada~a.', 1!. I. (Iq77L r..flccl o f :,train and dgc oll the binding o1" 7.12-dilnclh.,,lbenzl.,Janlhracenc (DM BAt to rat mammar.~ epithelial cell macromoleculc~,. Prec...Ira..-I.~. ( ' a m o r Rc~. 18, 20b4. King J.. Nons ('. R. & Bo~man M. ( ( 1 9 7 7 1 . [race anal)sis of dielh)lstilbestrol (I)I-S)in anilnal chtm. b,. parallel high-speed liqmd chrornalograph,., clcclroncaplure gas chromatograph.~, and radioassax,,../ ~hromat..~'~i. 15, 14 Kodell R. I... Haskin M. (i.. Shax~ (i. ~'. & (Ja}]ol l) ~" (1983). ( ' I I R O N I ( ' ; A SAN procedure Ibr statistical anal}sis el carcinogenesis studie,, .I .%'tatiw. (',,input..%'mml 16, 2S7. Kodell R. L . Sha~ (J. ~ & Johnson ..X M. {It.l?42i. N o n - p a r a m e t r i c join[ eMiln:.itors lbr disease re,~lstance and survival functions in sur~ixal-sacrilice cxperlnlelltS. Biometrics .'l,tl, 43. Mcranzc D. R.. Gruenstem M. & Shinlkin M. B. (l~Jlgg) Eft;act of age and sex on the dcveh,pnlcnl o f neopl:.isnl~, in Wistar rats recelxlng ~1 ",ingle Jntragastrlc instillation e l 7.12-dlilielh)lbeni(;.i)anlhracenc. f i l l . . / ( +mr cr 4, 4Sit Nagas,~lwa tl. <1983L Prokictin its it prointHcr el inlllal progression o f sptlntalleOLl,~ l n i l l l l l i l i l l V

IUlllt)F'~

In nllcc

and kick o f relationship to age. lah, .S'+t 3,t, 14"~1. Pete R.. Pike M. ('.. I)a.x N. I(. (h'a) R (i.. l e o P X.. Parish S.. Pete J.. Richardn S. & Wahrendorf .1. I I~,xlll. (iuidclines li~r simple, serlsiti~¢ significance toms Ior Calcinogenic effects in long-term allJnlal experiments. In :~Iono.l~raph.~ oil Ihe I:raluatum ,>t the ('ar~ i#mk,cn#~ RAk ,,I ('henucal~ to Iluman.~. .~uppl. 2. I , , t e term and .S'horl?UI'##I ,~'f'#'t'~'#lill~ .'l~%fll'~ /Or ('(l#'Ul#1o~Cll%; tl ('#'IIHfl] -[pprai~al, p. 311. Internatumal Agent) (or Research on Cancer. I.yon. Richardson I). M. 11973). Spontaneous illainlmar',, II.lllllll incidence ii1 ('311 IleJ Inlet..hix Nott.',,. No 413. Jackson LahorcltOry. Bar thither. ME. Sinha Y. N.. Selb', 1. W.. Lev~is \ .I. & %:anderlaan \%: P (It172). Studcs o1" prolaclin secretion i11 mice h\ cl htmlologous radioJnlnltlllOaSSil~. I:)*docrimdo.g.i 63, S()fl. Tagliabue A., Ruco I... Mc('o~ J. L.. Mehzer M. S & Herberman R. B. (1978). IHect of macrophagc migration factor on peritoneal exudat¢ cells oF (311 l i e n lind ('3H. IIeJ mice. In Ori~i,s