Large doses of adenovirus type 12 were injected intraperitoneally into adult hamsters, and development of tumors and other pathological findings were studied in comparison with those in hamsters injected when newborn. Doses of 38~47 TCID60 per gram body weight produced tumors in 3 of 12 hamsters injected at 37~57 days of age. A dose of 170 TCID60 per gram body weight produced tumors in one of 18 hamsters injected at 61~71 days of age, but in none of 18 hamsters injected at 147~174 days of age, while the same dose per gram body weight produced tumors in 24 of 26 hamsters injected when newborn. In hamsters injected at adult ages, the number of tumors per animal decreased and the latent period for tumor development became very long as
compared with those in hamsters injected when newborn. Regardless of the age at the time of injection, acute inflammatory change was observed in the peritoneum which later developed into various degrees of peritoneal adhesion. Adenovirus type 3 also induced the peritoneal adhesion. Histology of tumors was studied and discussed.</p

Iwata, Katsumi

Murakami, Sakae

Ogawa, Katsuo

Yabe, Yoshiro

Okayama University Scientific Achievement Repository

Acta Medica OkayamaVolume 20, Issue 4 1966 Article 1AUGUST 1966Effect of injection of adenovirus type 12 inadult hamstersYoshiro Yabe∗ Katsuo Ogawa†Katsumi Iwata‡ Sakae Murakami∗∗∗Okayama University,†Okayama University,‡Okayama University,∗∗Okayama University,Copyright c©1999 OKAYAMA UNIVERSITY MEDICAL SCHOOL. All rights reserved.Effect of injection of adenovirus type 12 inadult hamsters∗Yoshiro Yabe, Katsuo Ogawa, Katsumi Iwata, and Sakae MurakamiAbstractLarge doses of adenovirus type 12 were injected intraperitoneally into adult hamsters, anddevelopment of tumors and other pathological findings were studied in comparison with thosein hamsters injected when newborn. Doses of 38∼47 TCID60 per gram body weight producedtumors in 3 of 12 hamsters injected at 37∼57 days of age. A dose of 170 TCID60 per gram bodyweight produced tumors in one of 18 hamsters injected at 61∼71 days of age, but in none of 18hamsters injected at 147∼174 days of age, while the same dose per gram body weight producedtumors in 24 of 26 hamsters injected when newborn. In hamsters injected at adult ages, the numberof tumors per animal decreased and the latent period for tumor development became very long ascompared with those in hamsters injected when newborn. Regardless of the age at the time ofinjection, acute inflammatory change was observed in the peritoneum which later developed intovarious degrees of peritoneal adhesion. Adenovirus type 3 also induced the peritoneal adhesion.Histology of tumors was studied and discussed.∗PMID: 4226975 [PubMed - indexed for MEDLINE] Copyright c©OKAYAMA UNIVERSITYMEDICAL SCHOOLActa Med. Okayama 20, 147-154 (1966)EFFECT OF INJECTION OF ADENOVIRUS TYPE 12IN ADULT HAMSTERS*Yoshiro YABE**, Katsuo OGAWA***, Katsumi IWATA*** andSakae MURAKAMI****Institute for Cancer Research**, Second Department of Pathology***and Department of Microbiology****, Okayama University MedicalSchool, Okayama, JapanReceived for publication, May 19, 1966The age of animals is a very important consideration in tumor virus ex-periments. Human adenovirus type 12 induced tumors in hamsters wheninjected at birth or at very young ages, but did not when injected at adultages1.2. In these experiments, tumors mostly developed at the site of injectionsuggesting that the local concentration of virus might be one of the importantfactors for tumor inductions. In the present experiment, large doses of adeno-virus type 12 were injected into adult hamsters and the development of tumorsand of other pathological changes has been studied.MATERIALS AND METHODSVirus: Adenovirus type 12, strain "Huie" was obtained from theAmerican Type Culture Collection and adenovirus type 3, strain" G. B." wasobtained from the Institute for Infectious Diseases, Tokyo University. Viruswas cultured in HeLa cells. The virus culture fl uid was frozen and thawed 3times, centrifuged at 1, 000 rpm for 5 minutes, and its supernatant was usedfor experiment.Virus titration: Virus fluid was diluted in tenfold series with Earlesolution containing 0.5% lactalbumin hydrolyzate and 0.1?-6 yeast extract(YLE). One-tenth milliliter of each virus dilution was inoculated into HeLacell tubes (4 tubes for each dilution) containing 1 ml of YLE as the maintenancemedium. These tubes were incubated at 36. 5°C and observed for cytopatho-genic effect for 5 days. The 50% tissue culture infectious dose per 0.1 ml(TCIDoo/O.l ml) was calculated by REED and MUENCH'S method4•Animal: The breeding colony was established in our laboratory withSyrian hamsters from the Medical Section of Fukuyama Detachment of Japan*This work was supported by a grant CA-06813 from the National Cancer Institute, U. S.Public Health Service, and by grants from the Ministry of Education of Japan.1471Yabe et al.: Effect of injection of adenovirus type 12 in adult hamstersProduced by The Berkeley Electronic Press, 1966148 Y. YABE, K. OGAWA, K. IWATA and S. MURAKAMISelf Defense Forces and from Fujii Farm, Kobe. Only the hamsters bred inthis colony were used in the present experiment. Virus was injected intraperi-toneally.Preparation of tissue sections: Tissues were fixed in formalin, andsections were stained with hematoxylin-eosin and silver impregnatio~.RESULTSDevelopment of tumors and peritoneal adhesion: As shown in Table 1,doses of 38--47 TCID60 per gram body weight produced tumors in 2 of 6Table 1 Development of Tumors and Peritoneal Adhesion in Hamsters IntraperitoneallyInjected with Adenovirus Type 12 at Various AgesAverage Virus dose injected No. of.A.ge .at body Virus titer I No. of No. of hamstersVirus InJectlon weight (TCID50/ I TCID/ injected hamsters withat I gram developing(days) injection O.lml)Iml iTCID body hamsters' tumors peritoneal(grams) I weightadhesionType 12 1-2I2.8 103.5 I 0.0151 480 171I26 24(31.107)b! 26II 37 68 III 0.1 I 3200 47 6 21100.264)! 01/ 51-57 1 85 II 0.1 1 3200 38 6 1(393) 0II 61-71 94 II 0.5 116000 170 18 1(112) 6II 147-174 132 I 1/ 0.7 122400 170 18 0 15Type 3 222-238 116 I II 0.7 22400 193 10 0 10cControl 1-2 2.8 I 0.015: I 13 0 0fluidI 0.5 ' III 61-71 9! 14 0 01/ 222-238 116 0.7 I I 10 1 0 0a: Number of injected hamsters surviving over 21 days after injection, b: Days fron injectionto death or sacrifice from tumors, given in parentheses, c: The peritoneal adhesion in hamstersinjected with type 3 was more marked than in those injected with type 12.All animals were observed for 23-563 days from injection to death or sacrifice.hamsters injected at 37 days of age, and in one of 6 injected at 51--57 days ofFig. lOne of hamsters intraperitoneally injected with adenovirus type 12 (170 TCID50/grambody weight) at 147-174 days of age and killed without tumors 222 days after injection. Fibrinousadhesion is observed among liver lobes, gallbladder and diaphragm. xlFig. 2 One of hamsters intraperitoneally injected with adenovirus type 3 (193 TCID50/grambody weight) at 222-238 days of age and killed 65 days after injection. Marked adhesion is ob-served among liver, stomach, intestine and diaphragm. XlFig. 3 Tumors at the retrorectal region of one of hamsters intraperitoneally injected withadenovirus type 12 (170 TCID50/gram body weight) at 61-71 days of age and dead 112 daysafter injection. X1/2Fig. 4 Spindle-shaped cells are arranged almost parallel to each other and attach to the finestroma resembling cycad leaves. On~ of common patterns. X4002Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/1Injection of Adenovirus 12 into Adult Hamsters 1493Yabe et al.: Effect of injection of adenovirus type 12 in adult hamstersProduced by The Berkeley Electronic Press, 1966150 Y. YABE, K. OGAWA, K. IWATA and S. MURAKAMi4Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/1Injection of Adenovirus 12 into Adult Hamsters 151age. A dose of 170 TCID50 per gram body weight produced tumors in one of18 hamsters injected at 61--71 days of age, while the same dose per gram bodyweight produced tumors in 24 of 26 hamsters injected within 2 days after birth.In hamsters injected at adult or young adult ages, one or two tumorsdeveloped in the abdomen in 100--393 days after injection. In one of thesetumorous hamsters, besides a colossal abdominal tumor, several (probably meta-static) tumors were also observed in the liver. In hamsters injected when new·born, multiple tumors developed in the abdomen in 31--107 days after injection.In many hamsters injected with 170 TCIDM per gram body weight, regard-less of age at the time of injection or of tumor development, various degrees ofadhesion of the peritoneum including the liver capsule were macroscopicallyobserved; liver lobes were adhering to each other, to the diaphragm, theintestine or the kidney (Fig. 1). The peritoneal adhesion was also observed inhamsters injected with adenovirus type 3, and the adhesion was more markedthan in those inje:::ted with nearly the same dose of type 12 (Fig. 2). None ofthe hamsters similarly injected with the control tissue culture fluid developedtumors or the peritoneal adhesion.Histo-morphological appearance of tumors and peritoneum: All tumorswhich developed in hamsters injected at 1--57 days of age, were histo-morpho-logically similar to the adenovirus-type-12-induced tumors developing in hamstersinjected at birth which had been reported previously5. In sections of thesetumors, tumor cells were usually spindle- or polygonal-shaped and packedclosely to each other, but with narrow artificial spaces among them suggestinga behavior of epithlial cells. The cell arrangement appeared mostly irregular,but, in places, spindle-shaped cells took palisade arrangement or rosette-likestructure. The spindle-shaped cells, and even the polygonal cells atached to thestroma with their extending cytoplasmic process (Figs. 4--6). There wereobserved only a few argyrophil fibers among tumor cells. On the other hand,tumors, which developed 112 days after injection at the retrorectal region ofone of hamsters injected at 61--71 days of age, resembled histologically lympho-sarcoma in appearance (Figs. 3, 7). The nuclei of these tumor cells weremostly spherical and the cytoplasm was scanty. In some parts, however. aFig. 5 A characteristic cell arrangement of the adenovirus-12-induced tumors showing apalisade pattern. Giant cells are observed among spindle-shaped cells. One of common patterns.x400Fig. 6 Tumor cells are large and polygonal in shape. One of common patterns. x400Fig. 7 Round-shaped cells with scanty cytoplasm are packed disorderly resembling lympho-sarcoma-like neoplasm. Two giant cells are observed. A section of tumors in Fig. 3. X 500Fig. 8 Several cells have a fine cytoplasmi.: process extending to the stroma. A section oftumors in Fig. 3. X 4005Yabe et al.: Effect of injection of adenovirus type 12 in adult hamstersProduced by The Berkeley Electronic Press, 1966152 Y. YABE, K. OGAWA, K. IWATA and S. MURAKAMismall number of cells attached to the stroma with their extending cytoplasmicprocess (Fig. 8). Numerous multinuclear giant cells were observed in all typesof tumors which developed in the present experiment.Two of hamsters injected with adenovirus type 12 at 61--71 days of agewere sacrificed 6 days after injection. The histological observation of the peri-toneum of these animals revealed the degeneration of serosa cells, the infiltrationof leucocytes and the proliferation of fibroblasts. In hamsters injected with thisvirus when newborn, similar findings were observed and reported previouslyll.In none of two hamsters similarly injected with the control tissue culture fluidand sacrificed, such findings were observed.Development of tumors and peritoneal adhesion in breeder hamsters:Thus far, 97 and 111 untreated hamsters in the breeder room have been ob-served for 12--28 and 6--12 months respectively. Of these, none have deve-loped tumors or the peritoneal adhesion.DISCUSSIONIn our previous work, adenovirus type 12 induced tumors only in hamtersyounger than 2 weeks of age2• In the present experiment, tumors were inducedeven in hamsters as old as 2 months of age by increasing the virus dose. But,in hamsters injected at adult ages, the tumor incidence and the number oftumors per animal decreased and the latent period for tumor development be-came very long compared with those in hamsters injected when newborn. Onthe other hand, the peritoneal adhesion, which is obviously the result of earlyinflammatory change of the peritoneum, was observed regardless of the age ofhamsters at the time of injection. All these results suggest that adenovirus type12 more or less produces inflammatory change-the early non-fatal manifestationof its infection-in both newborn and adult hamsters, but usually producestumors-the late fatal manifestation of its infection-only in those injected atvery young ages. Isolations of adenovirus type 12 from children with acuteinflammatory symptoms-the early manifestation of its infection-have beenreported6- 8• But presently nothing is known about the late manifestation of itsinfection in man. Our serological study have suggested that man, except forsome 3% of them, has little chance to be severely infected with adenovirus type12 within 6 months after birth9•As reported previously, our histo-morphological study of the hamstertumors induced by adenovirus type 12 suggested that the tumors would haveoriginated from neuroectodermal supporting cellsD• In the present experiment,tumors which developed in one of hamsters injected at adult ages, were mostlycomposed of round or polygonal cells resembling lymphocytic cells. Develop-6Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/1Injection of ,Adenovirus 12 into Adult Hamsters 153ment of a tumor resembling lymphosarcoma had been also reported in one ofhamsters injected intravenously with adenovirus type 12 at births. But therelation of some spindle-shaped cells to the stroma would suggest the possibilitythat this type of tumors might be also one of the neoplastic manifestations ofprimitive neuroectodermal, possibly neuroblastic, cells.SUMMARYLarge doses of adenovirus type 12 were injected intraperitoneally into adulthamsters, and development of tumors and other pathological findings werestudied in comparison with those in hamsters injected when newborn. Doses of38--47 TCID60 per gram body weight produced tumors in 3 of 12 hamstersinjected at 37--57 days of age. A dose of 170 TCID60 per gram body weightproduced tumors in one of 18 hamsters injected at 61--71 days of age, but innone of 18 hamsters injected at 147--174 days of age, while the same dose pergram body weight produced tumors in 24 of 26 hamsters injected when new-born. In hamsters injected at adult ages, the number of tumors per animaldecreased and the latent period for tumor development became very long ascompared with those in hamsters injected when newborn. Regardless of theage at the time of injection, acute inflammatory change was observed in theperitoneum which later developed into various degrees of peritoneal adhesion.Adenovirus type 3 also induced the peritoneal adhesion. Histology of tumorswas studied and discussed.REFERENCES1. TRENTIN, J. J., YABE, Y. and TAYLOR, G.: The quest for human cancer viruses. Science137, 835, 19622. YABE, Y., TRENTIN, J. J. and TAYLOR, G.: Cancer induction in hamsters by human type12 adenovirus. Effect of age and of virus dose. Proc. Soc. Exp. Bioi. Med. 111, 343,19623. YABE, Y., SAMPER, L., TAYLOR, G. and TRENTIN, J. J.: Cancer induction in hamsters byhuman type 12 adenovirus. Effect of route of injection. Proc. Soc. Exp. Bioi. Med. 113,221, 19634. REED, L.]. and MUENCH, H.: A simple method of estimating fifty per cent endpoints. Am.]. Hyg. 27, 493, 19385. OGAWA, K., TSUTSUMI, A., IWATA, K., FUJII, Y., OHMORI, M., TAGUCHI, K. and YABE,Y.: Histogenesis of malignant neoplasm induced by adenovirus type 12. Gann 57, 43, 19666. KIBRICK, S., MELENEDEZ, L. and ENDERS, J. F.: Clinical association of enteric viruses withparticular reference to agents exhibiting properties of the ECHO group. Ann. N. Y. Acad.Sci. 67, 311, 19577. PEREIRA, M. S. and MACCALLUM, F. 0.: Infection with adenovirus type 12. Lancet i, 198,19647Yabe et al.: Effect of injection of adenovirus type 12 in adult hamstersProduced by The Berkeley Electronic Press, 1966154 Y. YABE, K. OGAWA, K. IWATA and S. MURAKAMI8. OLSON, L. C., MILLER, G. and HANSHAW, J. B.: Acute infectious lymphocytosis presentingas a pertussis-like illness: its association with adenovirus type 12. Lancet i, 200, 19649 YABE, Y.: Antibody to adenovirus type 12 among infants. Lancet i, 1447, 19648Acta Medica Okayama, Vol. 20 [1966], Iss. 4, Art. 1http://escholarship.lib.okayama-u.ac.jp/amo/vol20/iss4/1

Effect of injection of adenovirus type 12 in adult hamsters

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