Induction of Intestinal Carcinoma in the Mouse byWhole-Body Fast-Neutron Irradiation*

PETER C. NowELL,f LEONARDJ. COLE, ANDMARIE E. ELLIS

(Division of Biological and Medical Sciences, U.S. Naral Radiological Defense Laboratory, San Francisco £4,Calif.)

Tumors of the intestinal tract in rats and miceare rare. In one large series reported by Wellset al. (17), only nineteen primary tumors of thebowel were found in 142,000 mouse autopsies; ofthese, eleven occurred in prolapsed rectums andonly one in the cecum. No tumors were found inthe small intestine. Even after whole-body exposureto various kinds of ionizing radiations includingx-rays, gamma-rays, and fast and slow neutrons,mice exhibit an intestinal tumor incidence of lessthan 1 percent (11-12,16). Significant numbers ofintestinal neoplasms have been reported previously only in rats after very high doses of radiation tothe bowel mucosa, produced either by feedingradioactive yttrium (13), by localized irradiationwith 190 Mev deuterons (3), or by lethal doses ofwhole-body x-radiation followed by parabiosispostprotection (4). Only in the latter group didthe tumors metastasize. The observation, reportedhere, of a high incidence of metastasizing intestinaltumors in mice exposed to sublethal, whole-bodyfast-neutron irradiation is, therefore, of considerable interest.

MATERIALS AND METHODSIn several separate experiments, 194 male and female

LAFi mice (4-5 months old) were irradiated in a single whole-body exposure with fast neutrons in the 60-inch cyclotron atthe Crocker Laboratory, University of California, Berkeley.For each exposure twelve mice were placed individually inthin-walled lusteroid centrifuge tubes placed radially on aí-inchthick, 15-inch diameter wooden disk. Several air holeswere drilled in each of the lusteroid tubes, which were closedwith cork stoppers during irradiation. Details of the exposuregeometry and physical set-up for neutron irradiation have beendescribed in a previous report by Carter et al. (6).

The fast-neutron beam from the 60-inch Crocker Labora-

* This work was supported, in part, by funds from theBureau of Medicine and Surgery, U.S. Navy Department.The opinions and assertions contained herein are the privateones of the authors and are not to be construed as official or asreflecting the views of the Navy Department.

Presented, in part, at the 47th Annual Meeting of theAmerican Association for Cancer Research, Atlantic City,New Jersey, April 13-15, 1956.

t Lt. (MC) U.S.N.R.

Received for publication May 1, 1956.

tory cyclotron was obtained with 20 Mev deuterons, with theBe(D,n)B reaction. Neutron flux and spectral measurementswere carried out by the Radiological Physics staff of thislaboratory with sulfur threshold detectors and nuclear trackplates. The physical characteristics and dosimetry considerations of cyclotron neutrons have been described in a report bythis same group (Tochilin et al. [14]). The mean neutron energyof this beam was 8 Mev, with 93 per cent of the neutronspossessing energies above 3 Mev. At the mouse exposure position 30 inches from the target, the neutron output was approximately 80 rep per minute (according to the conversionfactor of 1.8 X IO8sulfur-neutrons per rep). The total neutrondose received ranged from 290 to 580 rep, plus a gamma-raycontamination component equal to approximately 5 per centof the neutron dose.

An additional smaller group of 28 LAFi mice was exposedto 2 Mev neutrons, obtained by the Be(p,n)B reaction, with 12Mev protons in the cyclotron. The gamma-ray contaminationof this beam was equivalent, approximately, to 15 per cent ofthe neutron dose (15). The mice in this group received a neutron dose of 410-460 rep plus 60-70 r of gamma-radiation.

None of the mice exposed to 8 Mev neutrons died acutely,i.e., within the 30-day period after exposure. Of the 28 miceirradiated with 2 Mev neutrons, six died in less than 30 days(LDjo dose).

Following irradiation, all animals were caged as before,eight to ten per cage, and maintained in an air-conditionedmouse room. The mice received the routine laboratory diet(Purina Laboratory Chow) and water ad libitum. The animalswere observed at regular intervals and followed until death orsacrificed when obviously moribund. At autopsy, representative sections were taken from the various organs, fixed in 15 percent formalin, processed routinely, and stained with hema-toxylin and eosin. Mayer's mucicarmine stain was also used

when appropriate.Two populations of LAFi mice were utilized for compaiison

with the neutron-irradiated mice. The first was a group of 658nonirradiated controls, which were maintained under the sameconditions and environment as the neutron-irradiated mice. Todate 165 mice of this group have been examined at death,when 12-28 months old. The second group comprised 136 micewhich had survived a single supralethal dose (800 r as measured in air) of whole-body x-radiation (250 kvP), by means ofpostirradiation treatment with a single intraperitoneal injection of LAFi mouse spleen homogenate, as described previously in reports from this laboratory (7, 8). A detailed presentation and analysis of this population of mice, in terms of incidence of neoplastic and non-neoplastic lesions are being reported separately (9). The age at death of the mice in thisgroup ranged from 4 to 24 months.

RESULTSIntestinal neoplasms.—To date, 13-16 months

after radiation exposure, 63 neutron-irradiated

873

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874 Cancer Research

mice have been examined. The incidence of intestinal tumors is shown in Table 1. Of the seventeen intestinal neoplasms, eleven were in thececum and six in the jejunum or ileum (Figs. 1 and2). The first tumor appeared 7 months after irradiation, and there has been no sex difference inincidence. All tumors showed extensive local invasion; many had metastasi/ed to the regionallymph nodes: and some had caused death byperforation and peritonitis.

Histologically (Figs. 3-7), most of the tumorsexhibited considerable variation in cell type, fromrelatively well formed neoplastic glands to sheetsof completely undifferentiated "signet-ring" cells.

In many cases, the same tumor showed these

TABLE1INCIDENCEOFINTESTINALCARCINOMAINMICE

Type of No. No. Per centradiation mice tumori tumor«

Neutron—8 Mev SS 12 34410-580 rep

Neutron—8 Mev 16 O O290-350 rep

Neutron—2 Mev 12 5 42410-460 rep

TotalX-ray+spleen

homogenate(790-810 r)

Nonirradiated

68*

186

165

17 27

* No. of mice which have died to date, 16 months afterirradiation.

extremes of differentiation, and in all cases mucusproduction was extensive. In the better differentiated portions of the carcinomas of the smallintestine, recognizable Paneth cells with secretorygranules were noted. Besides invasion of the bowelwall and lymph node métastases,occasional tumors showed microscopic evidence of extensioninto adjacent organs including the pancreas,epididymis, and adrenal.

Examination of sections of the cecum andsmall bowel from neutron-irradiated mice dyingwithout intestinal tumors showed mucosal atrophywith focal areas of hyperplasia in some of the micereceiving the higher dosages. In others, however,no histologie abnormalities at all were noted. Norwas there seen in any case, with or without tumor,evidence of the extensive scarring and "pinching-off" of normal intestinal epithelium, such as ap

parently preceded intestinal tumors in the ratsgiven localized deuteron irradiation (3).

In one mouse with severe atrophie changes ofthe cecal mucosa, several foci of hyperplasiashowed sufficient cellular atypicality and invasionof the submucosa to warrant classification as early

adenocarcinoma. This observation may implythat the cecal atrophy represents a premalignantchange.

No intestinal tumors were found in either thenonirradiated mice or in the x-radiated micetreated with spleen homogenate (Table 1), nor, inthese groups, was any evidence noted of thesevere atrophie and hyperplastic mucosal changesobserved in some of the cyclotron-irradiated mice.

It is of interest that in twelve of the neutron-irradiated mice, both with and without intestinaltumors, hyperplastic changes were found in themucosa of the pyloric and prepyloric portions ofthe stomach, with distorted overgrowth of gastricglands and some cellular atypism. Thus far, onlyone of these lesions has shown invasion or otherdefinitely malignant characteristics. None of thenonirradiated or x-radiated mice has shownidentical lesions, although one of the former groupdeveloped a squamous-cell carcinoma of thestomach, and two of the latter group showedglandular hyperplasia in conjunction with severegastric amyloidosis.

Lesions in other organs.—Inthe neutron-irradiated mice, the increase in incidence of lesions outside of the intestinal tract has not been so strikingas that of the intestinal tumors. Thus, nonin-testinal malignancies, ovarian adenomas, andsclerotic changes in the kidneys have been morecommon than in nonirradiated controls of thesame age, but these lesions have not been so frequent as in the supralethally x-radiated (800 r)mice treated with spleen homogenate. Except forthe intestinal tumors, therefore, pathologicchanges in the neutron-irradiated mice have beensimilar to what is seen experimentally following acomparable, i.e., sublethal dose of x-rays (11).

Longevity.—Themortality data are presented inChart 1. It can be seen that the neutron-irradiatedmice have, to date, died at the same rate as thex-radiated mice, with the latter reaching 50 percent mortality approximately 8 months earlierthan the nonirradiated control population.

The mortality curve for the x-radiated mice isbased on 295 LAFi mice protected against 800 r byspleen homogenate in experiments performedseparately from those affording the tumor incidence data (9).

DISCUSSIONThe action of fast neutrons in producing in

testinal tumors in LAFi mice is not readily explained. Either it represents a specific qualitativeeffect of the neutrons on the intestinal mucosa orit is the result of a relatively greater dose of radiation at this site. It has been postulated previously

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NOWELLe<al.—Neutron Induction of Mouse Intestinal Carcinoma 875

(5) that, in total-body neutron irradiation, thebowel mucosa may actually receive a higher dosethan the rest of the body. This suggestion is supported by Bloom's observation (2) that there is an"apparent" quantitative difference between fastneutron and x-ray effects on the bowel. In thislaboratory, mice were sacrificed 3 days after receiving either 450 rep of fast neutrons or 450 r of250 kvP x-rays, and examined histologically. Muchgreater mucosal damage was noted in the small intestine and cecum of the neutron-irradiated micethan in the x-radiated mice, while bone marrowdamage was not strikingly different in the twogroups. The report of Carter et al. (6) of an intestinal RBE (relative biological effectiveness) of2.3 for fast neutrons, as compared with 250 kvPx-rays, based on bowel weight changes, is furtherevidence for a differential action of neutrons onthe intestine. The RBE values for spleen and thymus weight were 1.7 and 1.5, respectively (6).

Brennan et al. (5) suggested that the apparentincrease in dose to the intestinal mucosa could bedue to the relatively high boron content of mousefood. Since boron has an extremely high cross section for thermal neutron capture, the reaction :

B10+ n -> [B11]-»Li7 + a (2.4 Mev)

could take place in the intestinal contents andthus through local alpha-irradiation effectively increase the dose to the intestinal epithelium. Sincethe flux of thermal neutrons at the depth of themouse intestine in the fast-neutron experimentsreported here was relatively very low (14), itseems unlikely that boron capture of slow neutrons could play a major role in the differentialeffects observed. However, studies aimed at resolving this question are under way.

Carter et al. (6) have suggested another possible explanation for the observed high intestinalRBE of neutrons. Since bone has a relatively lowhydrogen content, the dissipation of neutronenergy within it and adjacent marrow is low compared with that in the soft tissues. Thus, the bowelwould receive a relatively higher dose of irradiation than the hematopoietic tissues.

The induction of tumors, however, may not bedue simply to an increase in the acute intestinaldamage. Blair (1) has suggested that fast neutronsproduce greater irreparable damage than x-rays, sothat differences in long-term effects (i.e., cataracts,decreased longevity) may be relatively greaterthan short-term pathologic changes. However, except for intestinal tumors, in the present experiments there has been no striking increase in theincidence of long-term effects as compared with

effects in animals receiving comparable doses ofx-ray (11).

It is of interest that there does appear to be adefinite threshold dose, in the vicinity of 400 rep,for the induction of intestinal tumors, as shown inTable 1. If tumor incidence in the intestine issimply a function of radiation dose, and if theneutron RBE value of 2.3 is applicable, one mightthus anticipate the production of such tumors inmice surviving x-ray doses of 900-1,000 r. Attempts to obtain such experimental conditionsthrough postprotection by spleen and bone marrow preparations are being made. The finding, byBrecher et al. (4), of intestinal tumors in rats surviving lethal x-ray doses through postprotectionby parabiosis, lends support to this possibility.

TOTALCUMULATIVEMORTALITY(PERCENT—¿�fs>ol-bUi<T>-4CDU>CAOOOOOOOOOOCJ^A—

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2 4 6 8 IO lì 14 16 18 30 22 24 26 28 30 3<AGE AT DEATH (MONTHS)

CHARTl.—Cumulative mortality of mice exposed to fastneutrons or x-rays.

The fact that other workers have not observedintestinal tumors in mice exposed to lethal andsublethal doses of fast and slow neutrons (12, 16)is possibly owing to the high contamination of thebeam by 7-rays, so that only a fraction of the totaldose received by the animals was actually due toneutrons. Thus, this report is probably the firstlong-term study on mice receiving a high dose(290-580 rep) of relatively "pure" fast neutrons.

As yet, there is no information whether the induction of intestinal tumors by fast neutrons is astrain-specific phenomenon. It is of interest in thisconnection that, of four CFi mice surviving fast-neutron irradiation, one developed a very earlyadenocarcinoma of the cecum 17 months after irradiation, with invasion of the submucosa (Fig. 8).Neutron studies in another strain of mice (C57BL)are being carried out at the present time.

SUMMARYOf 194 LAFi mice exposed to sublethal (290-

580 rep), whole-body, fast-neutron irradiation,plus 28 mice receiving 410-460 rep of 2 Mev neu-

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876 Cancer Research

trons, 63 have died to date, 16 months followingirradiation. In 17 (27 per cent), intestinal carcinomas have been found. Eleven tumors werelocated in the cecum and six in the jejunum orileum. All have been mucoid adenocarcinomas,showing extensive local invasion and frequentmetastasis to regional lymph nodes.

No intestinal tumors were found in 165 non-irradiated LAFi mice or in 136 mice receiving"supralethal" doses of x-ray (800 r).

Fast neutrons appear to act differentially on theintestinal mucosa, both with respect to acutedamage and long-term effects. Other organs ofneutron-irradiated mice have not shown thispreferential sensitivity to neutrons in the development of tumors or other pathologic lesions.

ACKNOWLEDGMENTS

The authors wish to thank Dr. J. G. Hamilton, Director ofthe Crocker Laboratory, and the late Mr. G. B. Rossi formaking the cyclotron available, and acknowledge the cooperation of the operating staff.

The tissue sections were prepared and stained by Miss P. L.Roan and the staff of the Pathology Laboratory. The photomicrographs were made by Mr. Victor Duran.

REFERENCES

1. BLAIR,H. A. A Formulation of the Injury in Life Span,Dose Relations for Ionizing Radiations. 1. Application tothe Mouse. USAEC unclassified report, LR-206, 1952.

2. BLOOM,W. Histopathology of Irradiation from Externaland Internal Sources, p. 510. New York: McGraw-Hill,1948.

3. BOND,V. P.; SWIFT,M. N.; TOBIAS,C. A.; and BRECHER,G. Bowel Lesions Following Single Deuteron Irradiation.Fed. Proc., 11:408-9, 1952.

4. BHECHEH,G.; CRONKITE,E. P.; and PEERS, J. H. Neoplasms in Rats Protected against Lethal Doses of Irradiation by Parabiosis or Para-aminopropriophenone. J. Nat.Cancer Inst., 14:159-75, 1953.

5. BRENNAN,J. T.; HARRIS, P. S.; CARTER,R. E.; andLANGHAM,W. H. The Biological Effectiveness of ThermalNeutrons on Mice. Nucleonics, 12 (4): 31-35, 1954.

6. CARTER,R. E.; BOND,V. P.; and SEYMOUR,P. H. TheRelative Biological Effectiveness of Fast Neutrons inMice. USNRDL Report TR-20, 1954.

7. COLE,L. J., and ELLIS,M. E. Strain and Species Factors inPost-Irradiation Protection by Spleen Homogenates. Am.J. Physiol., 173:487-94, 1953.

8. COLE, L. J.; FISHLER,M. C.; ELLIS, M. E.; and BOND,V. P. Protection of Mice against X-Irradiation by SpleenHomogenates Administered after Exposure. Proc. Soc.Exper. Biol. & Med., 80:112-17, 1952.

9. COLJE,L. J.; NOWELL,P. C.; and ELLIS,M. E. Incidence ofNeoplasms in Mice Protected against Lethal Doses ofX-Rays by Spleen Homogenate. "Abstr.," Proc. Am.

Assoc. of Cancer Research, 2:100, 1956; J. Nat. CancerInst. (in press).

10. FURTH,J., and FUHTH,O. B. Neoplastic Diseases Producedin Mice by General Irradiation with X-Rays. I. Incidenceand Types of Neoplasms. Am. J. Cancer, 28:54-65, 1936.

11. FUHTH,J.; UPTON,A. C.; CHRISTENBERRY,K. W.; BENEDICT,W. H.; and MOSHEM,J. Some Late Effects in Miceof Ionizing Radiation from an Experimental Nuclear Detonation. Radiology, 66:562-69, 1954.

12. HENSHAW,P. S.; RILEY,E. R.; and STAPLETON,G. E. TheBiological Effect of Pile Radiations. Radiology, 49:349-60,1947.

13. Lisco, H.; BRUES,A. M.; FINKEL, M. P.; and GHUND-HAUSER,W. Carcinoma of the Colon in Rats Following theFeeding of Radioactive Yttrium. Cancer Research, 7:721-25, 1947.

14. TOCHHJN,E.; Ross, S. W.; SHUMWAY,B.; KOHLER,G. D.;and GOLDEN,R. Cyclotron Neutron and X-Ray Dosimetryfor Animal Irradiation Studies. Radiation Research, 4:158-73, 1956.

15. TOCHILIN,E.; SHUMWAY,B.; and KOHLER,G. D. Responseof Photographic Emulsions to Charged Particles. Radiation Research (in press).16. t'pTON, A. C.; FDRTH,J.; and CHRISTENBERHY,K. W.

Late Effects of Thermal Neutron Irradiation in Mice.Cancer Research, 14:682-90, 1954.

17. WELLS,H. G.; SLYE,M.; and HOLMES,H. F. ComparativePathology of Cancer of the Alimentary Canal, with Reporton Cases in Mice. Am. J. Cancer, 33:223-38, 1938.

(All sections stained with hematoxylin and eosin).Fio. 1.—Tumor in cecum of neutron-irradiated LAFi

mouse.FIG. 2.—Tumor in jejunum with dilation of obstructed

proximal loop.FIG. 3.—Adenocarcinoma invading wall of cecum. The sur

face of the tumor shows inflammation and necrosis. X75.FIG. 4.-—Adenocarcinomaof cecum. Note variation from

relatively well differentiated glands at top to undifferentiated"signet-ring" cells at bottom. X255.

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Fio. S.—Tumor cells within lymphatics in wall of cecum.X375.

FIG. 6.—Metasta tic adenocarcinoina of cecum in mesen-teric lymph node. X152.

FIG. 7.—Adenocarcinoinaof jejunum. Poorly differentiatedtumor cells replacing normal mucosa and invading adjacentpancreas. X152.

FIG. 8.—Earlyadenocarcinoma of cecum in neutron-irradiated CFi mouse. X152.

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1956;16:873-876. Cancer Res   Peter C. Nowell, Leonard J. Cole and Marie E. Ellis  Fast-Neutron IrradiationInduction of Intestinal Carcinoma in the Mouse by Whole-Body

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