Induction of foci of altered hepatocytes by a single injection of azaserine to rats

Cancer Letters, 7 ~'1979) 265--272 © Elsevier/North-Holland Scientific Publishers Ltd.

265

INDUCTION O F FOCI O F A L T E R E D H E P A T O C Y T E S BY A SINGLE INJF.CTION O F A Z A S E R I N E T O R A T S *

SEIIICHITAKAHASHI, S I K A N D A R L. K A T Y A L , BENITO L O M B A R D I aridHISASHI SHINOZUKA

Dep~rtment of P~thology, University of Pzttsburgh, School of Medmtne, Plt#sburgh, PA 15261 (U.S.A.)

(Received 12 February 1979) ( Rewsed versmn received 11 Ap~il 1979) (Accepted 16 April 1979)

SUMMARY

T h e induction o f loci o f altered, 7-glutamyltranspeptldase (GCT)-positlve h e p a t o c y t e s by azaserine was investigated. A f t e r injection o f a single dose of azaserine, m a n y loci developed in male Wistar rats fed a cholme-devozd'(CD) diet containing acetylaminofluorene (AAF), b u t only a few m ral,s fed a choline-supplemented (CS) diet containing A A F . Similar results were obtained in rats fed a plain CD diet or a plain CS diet a n d injected with a single dose o f azaserine after a partial h e p a t e c t o m y . These findings indicate :;hat azasefine is an effective in: ator of liver carcinogenesis in rats, a n d t h a t a CD dmt acts as a strong p r o m o t e r o f t h e evolution m i t m t e d liver cells to loci of altered, GGT-positlve hepatocytes.

INTRODUC~ON

A z ~ e r i n e (O-dlazoacetyl-L-serine) is a recently d~scovered chemical carcinogen w h m h possesses a high degree o f organ specificity [ 6 ] . II~ rats, it induces a high incidence of acinar cell a d e n o m a s ~md adenocarcino-nas of the pancreas, b u t 0nly a very low incidence o f k i d n e y mad hver t u m o r s after a long latent period [5,6]. A single rejection o f azaserine to rats ha~ been shown to cause DNA damage in several organs [ 4 ] . However, d e s p l ~ th~ difference Address all cor,~e~pondence to. Dr. H. Shinozuka, Department of Pathology, University of

P~ttsburgh, School of Medmine, Pittsburgh, PA 15261, U.S.A. * This ~tudy was supported in part by grant CA 23499 From the National Cancer Instltu~ and a ~rant from ~he Samuel and Emma Winters, Foundation. Abbreviations. AAF, 2-acetylaminofluorene; CD, choline devoid, CS, chohne supplemented; GGT, ~,-glutarnyltranspeptidase.

266

in organ susceptlbihty to azaserine carcinogenesis,no differences in the degree of D N A damage or rate of its repair were detected in pancreas, kidney and hver. It was suggested, therefore, that tumors develop in the liverwith a low incidence because of a lack of appropriate promotion in this organ [4]. In an earlierstudy [11], we demonstrated a st~'ikingenhancement of hepatoma Induction by azaserind in rats fed a C D diet, and suggested that the diet m a y have a promoting action on azaserine liver carcinogenesis. ]In the present study, we used Solt and Farber's procedure [14,15] for the rapid induction m rat liverof foci of altered, GGT-positive hepatocytes, to examine whether loci could be elicited,under the promoting action of a C D diet,by a single iz:Ljection of azasermc.

TABLE 1 DIET COMPOSITION (g/kg) CS Alcohol-extracted peanut meal a Water-washed soya protein a Alphacel b Corn starch Dextrin b Sucrose Casein, vitamin free b L-Cystine Salt mixture a,c Sucrose vitamin mixture a,d Choline chloride Corn oil-tocopherol c Corn oil Primex f To~l

CD

120.00 80.00 10.00 100.00 100.00 381.00 10.00 2.00 29.00 10.00 8.00 10.00 40.00 100.00

120.00 80.00 10.00 100.00 100.00 389.00 10 00 2.00 29.00

1000.00

1000.00

I0.00 0 10.00 40.00 100.00

a Teklad Test Diets, Madison, WI. b ICN, Cleveland, OH. c Composition of the salt mixture (g/kg). CaCO3, 121.2176; CaHOP 4, 336.73; K~HPO4, 284.9254; MgSO4, 68.0~L66, NaCI4, 155.4138, ferric citrate (26.7% F3), 31.0828, MnSO4 • H~O, 1.5622, ZnSO4 " 7H~O, 0.3626, CuSO4 • 5~-I~O,4144; If.I, 0.0052, NaF, 0.0052; AIK(SO4)2 " 12H~O, 0.0414; CoCl 2 • 6 H : 0 , 0 0052; Na~SlO~ " 9H20, 0.2072, Na~sO2, 0.0104. d Composition of the vitamin mixture (g/kg): thiamine HCI, 0 500, riboflavin, 0.250; pyridoxine HCI, 0.200; d-calcium pantothenate, 1.00; mcotinic acid (niacin), 1.00; folic acid, 0.050; bsotm, 0.030, menadione, 0.100,p-aminobenzoic acid, 10.00, iuosstol, 50.00; vitamin B-] 2 (crystalline), 0.003;sucrose (q.s.) 936.867. e Composition (g): cod liver oil concentrate (McKesson Laboratorias, Bridgeport, Conn.), 1.00, a-tocopheryl acetate, 1.20; corn oil 97.80. f Hydrogenated vegetable oil, Proctor and Gamble Co., Cincinnati, OH.

267 MATERIALS AND METHODS

Male Wista~- rats (Hilltop L a b o r a t o n e s , Scot,dale, PA) weighing 130--150 g, were m m n t a i n e d on laboratory c h o w (Ralston Purina Co., St. Lotus, MO) for at least 1 week before begining t h e exper£ments. CD and CS diets were prepared as indicated in Table 1. T h e relative irr,portance o f r~he components o f t h e diets has been disucssed [ 1 3 ] . When indicated, A A F (AIdr~ch Chemicals, Madison, WI) was added to t h e diets at a level o f 0.02%. Azaserine (Calbiochem., LaJolla, CA) dissolved in 0.9% NaC1 solution was injected mtraperitoneally. Reagents for t h e histochemcial staining o f G G T were obtained f r o m Polysciences, Inc. (Warrington, PA). T h e basic design o f t h e 2 e x p e r i m e n t s is show~l diagramatically m Fig. 1. In t h e first experiment, rats were rejected with a single dose o f azaserme (50 mg/kg) at 0 time while t h e y were fed laboratory chow. O n e week later, t h e y were divided into 2 groups, o n e o f which was fed a CD + A A F dmt, and t h e o t h e r a CS + A A F diet. Control groups were injected with a saline solution at 0 time, instead of azaserine, a n d were subsequently treated in the same m a n n e r as the experimental groups. Subgroups of rats were sacrificed 3--4 weeks after t h e beginning o f t h e experiments. In t h e second experiment, rats were subjected to a 2/~; h e p a t e c t o m y [3] and, 18 h later, were rejected with a single dose o f azaserine (20 mg/kg). A f t e r 1 week on laboratory chow, t h e animals were divided into 2 groups, a n d fed either a plair~ CD diet or a plain CS diet. Control groups were injected with azaserine without prior partial h e p a t e c t o m y . Subgroups o f rats were sacrificed 4--5 weeks after the

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C$ DIET

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268

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269 beginning of t h e experiment. T h e n u m b e r o f ammals sacrificed m each subgroup is indmated in 'Table 2. B o d y weights o f anamals at t h e start of experim e n t s , at the tame of the dietary switches, and at t h e tame o f sacrifice are re,~orded. T h e livers were resected ior histological a n d histochemical e x a m ' n a t i o n . T h e tissue was fixed ~n Stieve's solution and sections were stained wath hemot o x y l i n and eosm. For hlstochemacal staining o f GGT, blocks o f laver were quickly frozen, and 5-pro cryostat sections were c u t and ~fixed m acetone• T h e s e c t m n s were stained according to R u t e n b e r g et al [9] GGT-posltive h e p a t o c y t e s foci wit]h a diameter larger t h a n 75/~m were c o u n t e d , and the results were expressed as n u m b e r / c m 2 o f liver sections• Differences betwee~ m e a n s were evaluated statistmally by t h e S t u d e n t s t-test. RESULTS Table 2 summarizes the results o f t h e 2 experiments. A single dose .of azaserine of 50 m g / k g body weight prevented n o r m a l gains o f b o d y weight during t h e first week. However, t h e groups o f rats which received t h e CS + A A F or t h e CD + A A F diets s h o w e d a comparable rate o f gain in body weight. In e x p e r i m e n t II, no signifman,L differences an final b o d y weaght were obserzed between rats which receaved t h e CS or t h e CO diet• In Expert-

Fig. 2. A focus o f ~,-GGT-positive hepatocy~es in the hver o f a rat given a single injection o f azaserine (50 m g / k g ) and then fed a CD + A A F diet for 3 weeks Frozen section x 110. Fig. 3. A h e m a t o x y l l n and eosin-stained section o f the liver o f a rat treated as described m Fig. 1. × 1 1 0 .

270 ment I, feeding a CS + AAF diet, without prior admin,istration of azaserine, produced no loci of altered, GGT-positive he~patocytes, whereas a few loci were present in the liver of ra~s fed the CD + AAF diet for 3 weeks. Numerous, readily identifiable loci were induced in the liver of rats tTreated with azaserine. However, the number and size of the foci were significantly greater in the groups e, [ rats ~ed the CD + AAF diet than in those fed the CS + AAF diet. (Number: P < 0.05, size: P < 0.01, a~ t,he 4th week). Figures 2 and 3 illustrate the typical appearance, in histochemically and heraatoxylin and eosm stained sections, of the loci that develop,,d in rats given a single injection of azaserine followed by feeding a CD 1- AAF diet for 3 weeks. Feeding the CD + AAF and the CD diets induced fairly extensive fatty livers whether or not azaserine was administered. However, hepatocytes in the foci were conststently less fatty than surrounding hepatocytes (Fig. 3). In Experiment II, rm foct developed in the liver of rats given azasenne without prior partial hepatec~omy and fed the CD or the CS diet for 4 weeks. In contrast, when a~asenne was injected 18 h after a partial hepatectomy, 10.9 -+ 5.1 focl/cm 2 of liver section developed in rats fed tbe CD diet, whereas in rats fed the CS diet no more than 1 focus/cm 2 wa,~ present. DISCUSSION There is evidence that the process o f chemical carcinogenesis, at least in the skin and the liver, consists of 2 major, basic stages: (1) initiation of target cel]s, due to interaction of the carcinogen(s) with cell macromolecules; and (2) promotion ol" the evolution o f initiated cells to neoplastic cells [1,7, 8]. In previous com~mnications [10,12], evidence was presented to suggest that a CD diet, and a CD diet containing AAF are possible promoters of liver carcinogenesis in rats, Indeed, after administration to the animals of a single dose of a chemical c-'trcinogen, the d~ets were found to promote readily ~,he proliferation a~ad evolution of initiated cells to loci of altered, GGT-positive hepatocytes, which ~tre considered to be precursor lesions of hepatocellular carcinomas [15]. The results prese~ ~ed in this commumcat~on show clearly that administration o f a singJe dose of azase~.dneto either intact or partially hepatectomized rats causes initiation of target ce~ls in the liver. Indeed, when the animals were fed thereafter ~ CD diel, or a CD diet containing AAF, a significant n umber o£ loci of altered, GGT-positive hepatocytes developed (Table 2). Foct developed, but in m~tch smaller numbers, also in rats dosed with azaserine and fed the CS or CS + AAF diets, and m ra~s dosed with saline and fed the CD + AAF diet. These results are in accord with our previous findings [10, ] 2], mad provide fuz'Lher evidence of the promoting effect of a CD diet on liver carcinogenesis. Four weeks after administration of 20 mg azaserine/kg body wt, loci develo]ped when the carcinogen was injected 18 h after a partial hepatec~omy, but not when it was injec~,ed to intact rats (Table 2, exp. II). These results may be expl;dned on the basis of the suggestion, recen~,ly

271 made by Cayama et al, [ 2 ] , t h a t cullalar replication, following interaction of chemical carcinogens ~mth liver cell DNA, is an essential step m completing the initiation of target cells. Liija et al. [4] showed that a single injection o f azaserine ~o rats fed a standard laboratory diet causes DNA'damage, and therefore presumably iratiates target cells in pancreas, liver and kidrey. No difference was observed ir~ either the degree of DNA damage or the rate o f its repair m the different organs. Yet, administration of azaserine to rats I'ed a standard laboratory diet led, after 12--24 months~ to the development, in a high percentage of the animals, o f pancreas acinar cell carcinomas, whereas the incidence of liver and kidney t u m o r s was very low [6]. For this reason, azaserine has been used almost exclusively so far in studms concerned wLth the pathogenesis o f pancreas acinar cell carcinoma. However, administration o f azaserine t~ rats fed a CD diet resulted, wlthra 6 months, m the reduction o f hepatocellular carc.lnomas in 70% o f the animals [11]. Preneopl~tic nodules, but no carcinomas, developed within th~s time m the pancreas of She anim',L~" nodules were fewer than in rats dosed w~tb the same a m o t m t of a z a s e , , n e and fed a CS dmt. These findings clearly illt~strate that cancer promoting agents or factors, such as a CD diet, can play a.a unportant role m determining ~he primary site of t u m o r development, and that the primary site may be determined n o t as much by the chemicd na?.ure and partmular organotropism of the carcinogen, as by the nature and organotropism of the promoting agents or factors. REFERENCES 1 Berenblum, I. and Shublk, P (1947) The role of croton oil applications, associa~ed with a single paintm,~of a carcinogen, in turnout induction of the mouse's skin. Br. J.

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272 10 Set's, M . A . Katy.~l~ S.L., Sell, S., Shinozuka, H. and Lombardl, B. (1979) induction o f ~'oci of ~tcred ~ -glutamyltran~peptidase positive hepatocytcs in carcinogen treated rats fed a choline.devoid diet..~r. J. Cancer, in p'ress. 11 Shlnozuka, ~I., Katyal, S.L. and Lombardi, B. (~978) Azaserine c~treinogenesis: Organ susceptibil;ty c h ~ g e m rats fed a diet devoid of choline. Int. J. Cancer, 22, 86--3~9. 12 Sh~nozu~:a, H., S~:lls, M.A., Katy~d, S.L., Sell, S. ~nd Lombardi, B. Ef~eet~, of ~ cholinedev.~id diet on the emergence of ~.-glutamyltranepeptid.~e-positive loci in the liver of cazcinogen-|,reated raLs. Cancer Res., m press. 13 Shinozuka, H., L~rnl~,ardi, B., Sell, S. and Iamrn.~ino, R.M. (1978) Early histological and functionaJ al ~erations of ethJonine liver carcmngenesis in rats fed a chollne-defieient diet. Cancel' RerJ., 38, 1092--1098. 14 Solt, D.B. and Farber, E. (1976) A new principle for the sequential analysis of cbemreal carci~.ogenesis m,.~luding a quantitive assay ~'or ~ni~iation in liver. Nature, 263, 701--705 15 Solt, D.B., ~/ledllne, A. and Far~cr, E. (1977) Rapid emergence of carcino~en-induced h~perpl~tle lesions in a new model for the sequential analysis of liver carcinogenesis. A ~ . J. Pathol., 88, 595--618.