regeneration of syrian golden hamster pancreas and ... · 10 ml of insta-gel (packard instrument...

[CANCER RESEARCH 41, 1051-1057, March 1981]0008-5472/81 /0041-OOOOS02.00

Regeneration of Syrian Golden Hamster Pancreas and Covalent Binding ofA/-Nitroso-2,6-[3H]dimethylmorpholine1

Dante G. Scarpelli, M. Sambasiva Rao, Vadrevu Subbarao, and Marc Beversluis

Department of Pathology, Northwestern University Medical School. Chicago. Illinois 60611

ABSTRACT

The Syrian golden hamster is a potentially useful model forstudies of chemically induced pancreatic adenocarcinoma. Inan effort to refine the model so its sensitivity to carcinogen canbe enhanced such that dose and latent period are decreasedand tumor yield increased, experiments were undertaken toinduce pancreatic regeneration in the hamster. Random-bredmale hamsters given eight daily i.p. injections of DL-ethionine

(500 mg/kg body weight) in 0.9% NaCI solution while on amethionine-deficient diet developed pancreatic injury. This was

characterized by a progressive decrease (72%) in wet weightof pancreas accompanied by acinar cell atrophy and necrosis.Pancreatic regeneration initiated on the ninth day by a singlei.p. injection of L-methionine and return to a full amino acid diet

led to a gradual increase in wet weight of pancreas, reaching80% of control pancreas weight at 8 days later and full restitution by the 17th day. Mitotic activity, localized largely inacinar cells, rose from a total absence of mitoses on the eighthday before initiation of regeneration to a peak value of 16.8Â± 5.0 (S.E.) per 1000 cells 72 hr later, following which itdecreased steadily to a level of 2.8 Â± 1.5 per 1000 cells at192 hr. Islet and ductal epithelium showed peak indices of only1.8 Â±0.86 and 1.4 Â±0.6, respectively, at 72 hr. The mitoticindex of control animals was 0.84 Â± 0.76 per 1000 cells.Uptake of [3H]thymidine limited to acinar cell nuclei was evident

12 hr after initiation of regeneration (22 Â±5.5 per 1000 nuclei),reached a peak value of 224 Â±25 per 1000 nuclei at 60 hr,and decreased to 32 Â±26 per 1000 nuclei at 192 hr. Pancreatic slices from regenerating (60 hr) and normal pancreaswere incubated in vitro with 0.1 HIM A/-nitroso-2,6-[3H]di-

methylmorpholine for 90 min. Regenerating pancreas showedincreased covalent binding of carcinogen to DNA (21-fold),RNA (20-fold), and protein (3.7-fold) as compared to binding

observed in normal nonregenerating pancreas. In contrast tothe other rodent models of pancreatic regeneration, the hamster responds more rapidly and intensely, suggesting thatpancreatic regeneration may be a useful approach towarddevelopment of a limited dose schedule for pancreatic carci-nogenesis in this species.

INTRODUCTION

A number of /V-nitrosamines have been shown to be carci

nogenic for ductal epithelium of pancreas in the Syrian goldenhamster (34-36, 40). However, tumor induction is protracted,

requiring multiple doses of carcinogen administered for a minimum of about 20 weeks before significant numbers of tumorsare induced (26, 34). Such prolonged multiple-dose schedules

preclude detailed analysis of the initiation and promotion stagesof carcinogenesis. In the case of skin and liver, 2 widely studiedexperimental models of chemical carcinogenesis, considerableprogress toward elucidating some of the molecular eventsinvolved has been afforded by development of limited-dose

tumor induction schedules (1, 10, 11 ). These models make useof the fact that the sensitivity of target cells to a chemicalcarcinogen is greatly increased when they are replicating, i.e.,synthesizing DNA. In the case of skin, this is accomplished bytreatment with croton oil or its active principle, 12-O-tetra-decanoylphorbol-13-acetate (1, 17, 30-33), and in liver bysubtotal hepatectomy (7, 8, 21). A single-dose schedule for

induction of pancreatic ductal carcinoma developed by Pour efal. (37), using NBOP2 treatment of nonregenerating pancreas,

has a minimum latent period of approximately 39 weeks whichseverely limits its use for studies of initiation and promotion.The present paper reports the results of experiments aimedtoward the development of a limited-dose schedule for the

study of pancreatic carcinogenesis in the hamster using regenerating pancreas.

MATERIALS AND METHODS

Pancreatic Regeneration. Forty-eight postweanling (5 to 6weeks) male hamsters (Charles River Breeding Laboratories,Wilmington, Mass.), weighing 35 to 40 g, were maintained ona pelleted full amino acid semisynthetic diet (41) for 2 weeksprior to the start of the experiment, at which time they werechanged to a methionine-deficient diet. Simultaneously, theseanimals received daily i.p. injections of DL-ethionine (500 mg/kg body weight) in 0.9% NaCI solution for 8 days. Pancreaticregeneration was initiated on the ninth day by a single i.p.injection of L-methionine (800 mg/kg body weight), which wehave determined is equivalent to twice the average daily dietaryintake for hamsters of this age, and by returning the animals tothe full amino acid diet. Three experimental animals each weresacrificed on Days 0, 3, 5, 7, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 14, 15, and 17. Twenty-four hamsters maintained on

the full amino acid semisynthetic diet served as controls; 3animals each were sacrificed on Days 0, 3, 5, 9, 10, 11, 12,and 13 of the experiment. In an effort to test the growth-stimulatory effect of methionine, a second control group of 6hamsters was maintained on the full amino acid semisyntheticdiet and on the ninth day were given i.p. injections of L-methionine at the dosage mentioned above. Three animalswere sacrificed on Days 11.5 and 12. The pancreas wasremoved, weighed, and fixed in buffered formalin, and sectionsstained with hematoxylin and eosin were prepared for lightmicroscopic study. Quantitative estimation of regeneration was

1This work was supported in part by the Edith Patterson and Marie A. Fleming

Cancer Research Fund and the Cancer Research Fund, Northwestern University.Received July 21, 1980; accepted December 1. 1980.

2 The abbreviations used are: NBOP, N-nitrosobis(2-oxopropyl)amine; [3H]-dThd, pHJthymidine; [3H]NNDM. W-nitroso-2,6-(3H]dimethylmorpholine; RER.

rough-surfaced endoplasmic reticulum

MARCH 1981 1051

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D. G. Scarpelli et al.

accomplished by counting the number of mitoses in all phasesof division in 2000 randomly selected acinar, islet, and ductalcells per animal. The mitotic index was expressed as mitosesper 1000 cells. Several small fragments from each pancreas,measuring 0.5 to 1 mm, were fixed in cold 2.5% glutaraldehydein 0.1 M phosphate buffer (pH 7.5), embedded in Epon, sectioned on an LKB Ultratome (both as semithin sections for lightmicroscopy and as thin sections stained with uranyl acetateand lead citrate), and examined with a Hitachi HU-12B electron

microscope.Quantitation of DMA Synthesis during Regeneration. Ex

periments that were done to estimate the number of cells in Sphase of the mitotic cycle involved 31 hamsters in whichpancreatic necrosis had been induced as described previously.Nuclear labeling was accomplished by a single i.p. injection of[3H]dThd (1 Â¿iCi/gbody weight; specific activity, 2 Ci/mmol;

Research Products International Corp., Elk Grove Village, III.)at 12, 24, 36, 48, 60, 72, 96,144, and 192 hr after pancreaticregeneration was initiated by injection of methionine and returnto the full amino acid diet. Fourteen animals maintained on thefull amino acid diet since weaning served as controls; thesewere given injections of [3H]dThd at 12, 24, 48, 72, and 96 hr.Animals were sacrificed 90 min after injection of [3H]dThd, and

the pancreas was prepared for autoradiography. The numberof labeled nuclei per 1000 randomly selected acinar, ductular-

ductal, and islet cells was counted.Measurement of Covalent Binding of [3H]NNDM2 to DMA,

RNA, and Protein. In an effort to ascertain whether regenerating pancreas covalently binds carcinogen to a significantlygreater degree than does normal pancreas, in vitro experimentsutilizing the labeled /V-nitrosamine [3H]NNDM were undertaken.

The pancreas was removed from 8 hamsters 60 hr after theinitiation of regeneration, and surrounding fat and lymph nodeswere carefully dissected. The organs were sliced into 1- to 2-

mm lengths and placed in sterile Retri dishes containing 5 mlof sterile Krebs-Ringer bicarbonate buffer (pH 7.4) (gassedintermittently with 95% O2-5% CO2) and 50 juCi of [3H]NNDM

(0.1 HIM; specific activity, 53 mCi/mmol) kindly supplied by Dr.W. Lijinsky, National Cancer Institute-Frederick Cancer Research Center, Frederick, Md. Normal pancreas from 6 post-

weanling male hamsters maintained on the full amino acid dietand incubated under identical conditions served as controls.All incubations were continued for 90 min and were carried outwith gentle agitation of the Retri dishes. Following incubation,the slices were rinsed in 5 changes of cold Krebs-Ringer

bicarbonate buffer in which the last rinse contained no detectable radioactivity and homogenized in a Potter-Elvehjem ho-mogenizer containing 15 volumes of a 6% solution of p-ami-

nosalicylic acid. DNA, RNA, and protein were extracted with asolution containing 500 g of phenol, 70 g of m-cresol, and 0.5g of 8-hydroxyquinoline in 55 ml of water, according to amodification of the method of Kirby (23) as used by Hill ef al.(20). DNA and RNA were dissolved in 1 HIM phosphate buffer(pH 7.0), and protein was dissolved in 0.5 N NaOH. For scintillation counting, 0.5 ml of DNA, RNA, and protein was added to10 ml of Insta-Gel (Packard Instrument Co., Inc., DownersGrove, III.) and counted in a Beckman Model LS 9000 liquidscintillation spectrometer. DNA was determined by the methodof Burton (5); RNA content was quantitated by the method ofFleck and Munro (16). Protein content was measured by themethod of Lowry ef a/. (29) using serum albumin as standard.

RESULTS

Changes in Weight of Pancreas during Necrosis and Regeneration. As shown in Chart 1, maintenance of animals ona methionine-deficient diet and daily i.p. injection of ethionine

led to a progressive decrease in pancreas weight. On the ninthday of treatment, the average pancreas weight had decreasedto 27% of control pancreas weight. Regeneration was characterized by a rapid increase in pancreas weight which reached80% of control pancreas weight by the 17th day.

Mitotic Activity and Nuclear Labeling of Acinar Cells. Mitotic activity in regenerating pancreas was encountered mostfrequently in acinar cells; the mitotic index rose from an absence of mitoses on the eighth day before the initiation ofregeneration to a peak value of 16.8 Â±5.0 after 72 hr (p <0.05 when compared to control animals), and although it decreased steadily the index was still elevated (2.8 Â±1.5) at 192hr (Chart 2). A considerably lessened regenerative responsewas encountered in islet and ductal epithelium with peak indices of 1.8 Â±0.86 and 1.4 Â±0.6, respectively, at 72 hr. Mitoticindex of the first group of control animals maintained on a fullamino acid diet from the time of weaning was 0.84 Â±0.76.The mitotic index of the second group of hamsters in which L-

methionine was injected on the ninth day of the experimentwas 1.3 Â±0.3 seventy-two hr following injection.

Uptake of [3H]dThd was limited to nuclei of acinar cells (Fig.

1) (22 Â±5.5/1000 cells), was evident as soon as 12 hr afterinitiation of regeneration, reached a peak value of 224 Â±25/1000 cells at 60 hr, and decreased to 32 Â±26/1000 cells at192 hr (Chart 3).

120

Day

Chart 1. Percentage of change of wet weight of hamster pancreas duringdietary manipulations and administration of ou-ethionine followed by L-methionineas described in "Materials and Methods." Methionine-deficient diet and DL-

ethionine (â€¢)to Day 9, then a return to full amino acid diet plus u-methionine.Control animals were maintained on the full amino acid diet (O) throughout theexperiment. Points, average for 3 animals; oars, S.E.

1052 Pancreatic Regeneration and Carcinogen Binding



Pancreatic Regeneration and Carcinogen Binding

168 192

Hours

Chart 2. Mitotic index of pancreas during regeneration. Experimental group(â€¢)following initiation of regeneration by cessation of DL-ethionine treatment,administration of L-methionine, and return to the full amino acid diet. Control

animals (O) were maintained on the full amino acid diet throughout the experiment. Points, average for 3 animals; bars, S.E.

Binding of [3H]NNDM to Pancreatic DNA, RNA, and Protein.Covalent binding of [3H]NNDM to cell macromolecules in re

generating pancreas was increased 21-fold to DMA, 20-fold toRNA, and 3.7-fold to protein, as compared to binding observed

in nonregenerating pancreas. These were all highly significant(p < 0.001) (Table 1). Attempts to isolate highly purified DMAby CsCI gradient centrifugaron for the binding studies were notsuccessful due, presumably, to high intrinsic DNase activity inthe pancreas despite the addition of 4.2 mm EDTA to theincubation medium.

Morphological Alterations of Pancreas during Necrosisand Regeneration. After 4 days on the methionine-deficientdiet and daily injection of ethionine, extensive changes inacinar cells were evident. These consisted of a diminution ofcytoplasmic basophilia and zymogen granules and the presence of numerous cytoplasmic vacuoles. In every instance,ductular and islet cell epithelium appeared unchanged (Fig. 2).Varying degrees of acinar cell injury were encountered withinsingle acini, ranging from dilation of PER in cells which stillcontained zymogen granules to cells in which large vacuolesoccupying a major portion of the cytoplasm contained membrane fragments and dense bodies resembling zymogen granules (Fig. 3). Centroacinar, ductular, and ductal epithelium (Fig.4) showed no morphological evidence of injury. On the eighthday of ethionine treatment, the amount of acinar tissue wasgreatly diminished; individual acini were separated from oneanother by prominent trabeculae of loose connective tissuecontaining normal-appearing ducts and single cells (Fig. 5). In

250 -

2 200

Ooo

HoursChart 3. Kinetics of [3H]dThd labeling of acinar cell nuclei during regeneration

of pancreas. Experimental animals (â€¢)following initiation of regeneration bycessation of DL-ethionine treatment, administration of L-methionine, and return tothe full amino acid diet. Control animals (O) were maintained on the full aminoacid diet throughout the experiment. Points, average for 3 animals, unless shownotherwise; bars. S.E.

addition to the changes already described, the cytoplasm ofsome acinar cells showed a loss of the characteristic parallelarrays and fragmentation of RER (Fig. 6). Despite extensiveacinar cell injury, nests of atrophie acinar cells were present,devoid of zymogen granules, and with a surprisingly well-pre

served RER (Fig. 7). Cells in the interstitium were identified asacinar cells, fibroblasts, and macrophages. Twelve hr afterinjection of methionine and feeding the complete amino aciddiet, the Golgi membranes were readily apparent, and numerous vacuoles, some of which contained dense bodies, werepresent in the RER. A few zymogen granules and prominentmitochondria were also present. Sixty hr after regeneration hadbegun, almost all mitoses were localized in acinar cells, beingvery rarely present in ductal and islet cells and not at all in thevarious connective tissue cells in the interstitium (Fig. 8).

DISCUSSION

The necrogenic effects of ethionine on pancreatic acinarcells (3, 12, 19) and the remarkable capacity of injured pancreas to regenerate (13-15) have been the focus of much

work. Although this model has been well established in the rat,it is less so for other species (4, 28, 43, 44). In the hamster,studies have been largely limited to the Chinese hamster (3, 4)in which ethionine exerts an effect similar to that seen in therat (13-15) and guinea pig (44). Preliminary experiments with

MARCH 1981 1053



D. G. Scarpelli et al.

Table 1Covalent binding of I'HINNOM to DNA. RNA. and protein of normal and regenerating pancreas

Pancreatic slices were prepared from hamsters 60 hr after the initiation of regeneration as described in"Materials and Methods." Each experiment consisted of duplicate samples.

GroupControl

(nonregenerating)RegeneratingNo.

of experiments6

8Covalent

binding(dpm/mg)DMA

RNA16.4Â±14.1a 29.3 Â± 2.3

343.7 Â±97.3o 384.0 Â±99.3fcProtein42.2

Â± 3.1

159.5 Â±31Aba

Mean Â±S.E." p < 0.001.

ethionine in the Syrian golden hamster yielded erratic results;these were eliminated by placing the animals on a methionine-

deficient semisynthetic diet during administration of ethionine.Our results indicate that the Syrian golden hamster is an idealanimal model in which to study pancreatic regeneration, sinceit responds somewhat more acutely than does either the rat orguinea pig. Restitution of hamster pancreas began on Day 9and reached 80% of control weight 8 days later, as comparedto Day 10 and approximately 18 days for the rat (14). Further,labeling of acinar cell nuclei with [3H]dThd in the hamster

reached a peak value of 22.4% 60 hr after restitution began,as compared to 10% 72 hr after regeneration was initiated inthe rat (15). The brief and more intense regenerative responseobtained in the hamster may in part be attributable to the i.p.injection of L-methionine following cessation of ethionine treat

ment. In addition, the regenerative response in the hamsterappears to be much more intense in acinar cells in contrast tothe rat where ductal and islet cells, as well as interstitial cellsof possible mesenchymal derivation, seem to be activated to amore significant degree. A study dealing with the precursorcells involved during regeneration suggested that acinar cellsarise from surviving acinar cells and may also derive from ductepithelium, while the interstitial cells were assumed to beconnective tissue components of the repair process (13).

In the hamster, it appears that some acinar cells survive thenecrogenic effects of ethionine and are presumed to comprisethe cell population that responds by proliferation during regeneration of the pancreas. Although many single or small nests ofbasophilic cells were present in the interstitium of the hamsterat the height of the ethionine effect, electron microscopy identified the majority of these as atrophie, surviving acinar cells;although macrophages and fibroblasts were present, they constituted a minor proportion of the interstitial cell population. Nocells were identified which could be classified as undifferen-tiated and possibly constituting a so-called "stem cell" popu

lation during any period of regeneration. While this does noteliminate the possibility of such cells in the pancreas, theabsence of a morphologically recognizable population of un-differentiated cells, together with the fact that mitotic activityand [3H]dThd labeling are most intense in acinar cells during

regeneration, suggests that these cells are probably responsible for restitution of the pancreas.

The use of pancreatic regeneration and its attendant augmented cell replication to enhance the effects of pancreatropiccarcinogens in the rat has been reported previously (9, 24,25). In these experiments, partial pancreatectomy and ethionine were successful in increasing acinar cell tumorigenesis byazaserine (9, 24) and 4-hydroxyaminoquinoline 1-oxide (24,

25) in rats. However, to our knowledge, little or no informationis available concerning enhanced covalent binding of these

carcinogens to DNA of replicating pancreatic acinar cells inthis model. Binding of 4-hydroxyaminoquinoline 1-oxide to

DNA of regenerating rat pancreas is not increased over itsbinding to resting pancreas despite its enhanced tumorigenesiswhen administered as a single dose 3 days following theinduction of regeneration (24). This apparent anomalous resultremains to be elucidated. The increased covalent binding of[3H]NNDM to DNA and RNA, and to a lesser extent to protein,

as compared to nonregenerating normal pancreas in the experiments reported here, suggests that this may be a promisingmodel in which to study the early events of pancreatic carci-

nogenesis in the hamster. Covalent binding of a wide variety ofcarcinogens to DNA and other important macromolecules oftarget cells and tissues has been demonstrated to the extentthat it is generally accepted as a significant event in chemicalcarcinogenesis (22). Studies during the past few years haveshown that the molecular interactions of carcinogen with DNAinvolve strong bonds which lead to the formation of adductsthat are sufficiently stable that they are retained for prolongedperiods, so that lesions of DNA develop which may eventuatein alterations of gene expression (6). Before leaving this aspectof the discussion, it should be pointed out that, althoughcovalent binding of carcinogens to cell macromolecules maybe involved in carcinogenesis, the extent of its significanceremains to be established.

Utilization of the Syrian hamster model of pancreatic regeneration for carcinogenesis studies has an added advantage,since it may give some insight into the histogenesis of experimentally induced pancreatic carcinoma, in general, and thoseinduced by the 2-oxopropyl-containing nitrosamines, in particular. As it turns out, the origin of pancreatic ductal adenocar-

cinoma is currently a matter of controversy (39). On the onehand, there is evidence that this may arise as a result of acurious carcinogen-induced modulation of acini in which theyare converted to tubular or duct-like structures during carci

nogenesis; it is noteworthy that this change has been encountered in the genesis of pancreatic carcinoma induced by 7,12-dimethylbenz(a)anthracene in the rat (2), methylnitrosourea inthe guinea pig (38), azaserine in the rat (27), and NBOP in thehamster (42). In each instance, the duct-like component, which

closely resembles ductal adenocarcinoma, has been suggested to arise from dedifferentiated or otherwise altered acinarcells. On the other hand, Pour et al. (35) in their studies withthe hamster maintain that intra- and periinsular ductules are

the primary targets for the carcinogens. In support of acinarcell involvement in NBOP carcinogenesis is the fact that, in thehamster where the neoplasms induced are almost exclusivelyductal, the acinar cell appears to be more responsive to theacute and chronic effects of the carcinogen, as evidenced bynecrosis and later sustained increased mitotic activity (26, 42)

1054 CANCER RESEARCH VOL. 41



r_.-.- .,^,... -_,v

to a greater degree and earlier than seen in ductular and ductalepithelium. Some workers (26) have rationalized this discrepancy by suggesting that the mitogenic effect of W-nitrosobis(2-

hydroxypropyl)amine on acinar cells is due simply to regeneration subsequent to their necrosis. The role of acinar cells, ifany, in the pathogenesis of pancreatic ductal adenocarcinomaremains to be established and elucidated.

It will be of interest to see what the outcome of limited 2-oxopropyl-containing nitrosamine treatment, superimposed on

regenerating acinar cells, will be. If ductal carcinomas develop,then the notion of acinar cell modulation to ductal epitheliumduring the carcinogenic transformation must be seriously entertained. If, on the other hand, acinar cell tumors developexclusively, then ductal adenocarcinomas probably arise frompreexisting duct epithelium. A third possibility would be thatmixed ductal-acinar cell tumors arise. This would suggest,then, that both histogenetic possibilities exist.

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42. Scarpelli, D. G., and Rao, M. S. Pathogenesis of pancreatic carcinoma inhamsters induced by W-nitrosobis (2-oxopropyl)amine (BOP). Fed. Proc..37: 232, 1978.

43. Wachstein. M., and Meisel, E. Equal effectiveness of L and D-ethionine inproducing tissue damage in rats and mice. Proc. Soc. Exp. Biol. Med., 82:70-72, 1953.

44. Wenk, M. L.. Reddy. J. K., and Harris, C. C. Pancreatic regeneration causedby ethionine in the guinea pig. J. Nati. Cancer Inst, 52: 533-538, 1974.

MARCH 1981 1055



Fig. 1. Autoradiograph of hamster pancreas injected with a single pulse of [3H]dThd 60 hr after NBOP injection. Incorporation of [3H]dThd is limited to nuclei of

acinar cells. H & E, x 520.Fig. 2. Semithin (0.5 /im) Epon section of pancreas after 4 days on the methionine-deficient diet and daily injection of DL-etnionine. Extensive cytoplasmic

vacuolization and small aggregates of zymogen granules are evident in acinar cells. Ductular and ductal epithelium (arrows), and islet epithelium (open arrow) bycontrast appears free of vacuoles. Toluidine blue, x 580.

Fig. 3. Pancreatic acinus from pancreas shown in Fig. 2. Large vacuoles occupy a major portion of the cytoplasm of acinar cells, some containing membranefragments and others, cell debris (open arrow). Residual zymogen granules are present in one cell (Z), x 4500.

Fig. 4. Fourth day of ethionine-induced injury as in Fig. 2. A normal-appearing centroacinar cell (CAÃ•and ductular cells (D) are present adjacent to a necroticvacuolated acinar cell (A), x 6000.

1056



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Fig. 5. HistolÃ³gica! appearance of pancreas on the eighth day of ethionine-induced injury. There is a marked diminution of acinar tissue, and many of the smallclusters of cells in the interstitium are atrophie acini (arrows). Note the normal-appearing islet (/) and several ducts. H & E, x 380.

Fig. 6. Eighth day of ethionine-induced injury. Acinar cells showing cisternal swelling and fragmented PER. x 5900.

Fig. 7. Eighth day of ethionine-induced injury. Nests of acinar cells with well-preserved PER are devoid of zymogen granules. Note the surrounding basementmembrane, x 6600.

Fig. 8. Pancreatic acinus 60 hr following initiation of regeneration. PER consists of parallel arrays characteristic of normal acinar cells. A few zymogen granulesare present, both in the mitotic and in the resting acinar cells, x 5000.

1057



1981;41:1051-1057. Cancer Res Dante G. Scarpelli, M. Sambasiva Rao, Vadrevu Subbarao, et al.

H]dimethylmorpholine3-Nitroso-2,6-[NBinding of Regeneration of Syrian Golden Hamster Pancreas and Covalent

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