Toxicologic Pathology

Toxicologic Pathology http://tpx.sagepub.com/

Spontaneous Tumors in Aging (C57BL/6N X C3H/HeN)F1 (B6C3F1) Mice

Seiko Tamano, Akihiro Hagiwara, Masa-Aki Shibata, Yasushi Kurata, Shoji Fukushima and Nobuyuki Ito Toxicol Pathol 1988 16: 321 DOI: 10.1177/019262338801600302 The online version of this article can be found at: http://tpx.sagepub.com/content/16/3/321

Published by: http://www.sagepublications.com

On behalf of:

Society of Toxicologic Pathology

Additional services and information for Toxicologic Pathology can be found at: Email Alerts: http://tpx.sagepub.com/cgi/alerts Subscriptions: http://tpx.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://tpx.sagepub.com/content/16/3/321.refs.html

Downloaded from tpx.sagepub.com by guest on July 10, 2011

TOXICOLOGIC PATHOLOGY ISSN0192-6233 Copyright Q 1988 by the Society of Toxicologic Pathologists

Volume 16, Number 3, 1988 Printed in U S A .

Spontaneous Tumors in Aging (C57BL/6N X C3H/HeN)Fl (B6C3Fl) Mice* SEIKOTAMANO, AKIHIRO HAGIWARA, MASA-AKI SHIBATA, YASUSHI KURATA, SHOJIFUKUSHIMA, AND NOBUYUKI ITO First Department of Pathology, Nagoya City University Medical School, I Kawasioni, Mizuho-cho, Mizuho-ku. Nagoya 467, Japan ABSTRACT

Spontaneous tumors in untreated (C57BLJ6N x C3WHeN)FI (B6C3F1) mice used as controls in carcinogenicity tests were recorded. In both sexes, the development of spontaneous tumors was age-related. In 244 male mice, the most common tumors were hyperplastic nodules of the liver, hepatocellular carcinomas, malignant IymphomasAeukemias, lung adenomas, and adenocarcinomas. In 246 female mice, the most common tumors were malignant lymphomasfleukemias,pituitary adenomas, neoplastic nodules of the liver, hepatocellular carcinomas, and lung adenomas. Hepatocellular carcinomas metastasized in 20.3% of the animals with these tumors. Few other tumors except malignant lymphomas and leukemias metastasized. Various tumors of other organs and/or tissues were found at low incidences.

INTRODUCTION Data on spontaneous tumors in rodents provide useful background information for evaluating the results of carcinogenesis tests. Historical data on spontaneous neoplastic lesions in mice are of special significancein this respect. There are several reports of their incidences in (C57BL/6N x C3H/HeN)FI (B6C3FI) mice in the USA (2, 9). Since B6C3FI mice are now usually used in chronic toxicity and carcinogenicity studies in Japan, the present paper reports data on the incidences of neoplastic lesions in control B6C3FI mice in our laboratory. MATERIALS AND METHODS Weanling inbred male and female barrier-sustained B6C3F, mice used as controls in 5 carcinogenicity studies in our laboratory were obtained from Charles River Japan, Inc., Atsugi, Japan. Data on these 244 males and 246 female mice in the 5 control groups are presented in this report. The animals were housed 5 to a plastic cage with hardwood chips for bedding under similar conditions in each experiment (room temperature, 20 f 2°C; humidity, 55 & 10%;light: dark cycle, 12:12 hr) in a semibarrier-system animal room, and had free access to

* Address correspondenceto: Sciko Tamano, First Department of Pathology, Nagoya City University Medical School, 1 Kawasumi, Mituho-cho, Mizuho-ku, Nagoya 467, Japan. 32 1

commercial diet (Oriental M powdered diet, Oriental Yeast Co. Ltd., Tokyo, or CE-2 powdered diet, CLEA Japan Inc., Tokyo) and water. All survivors at the end of the 2-year experiments were subjected to complete necropsy. Mice that died or became moribund during the experiment were also autopsied. All gross lesions in each animal were recorded. Organs and tissues were fixed in 10% phosphatebuffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). The following organs and tissues were examined microscopically: the heart, aorta, lymph nodes (mandibular and mesenteric), spleen, bone with marrow (from the sternum and tibio-femoral joint), thymus (when present), pituitary, thyroid (with parathyroid), adrenals, nasal cavity, trachea, lungs (at least 2 lobes and the mainstem bronchi), tongue, salivary glands, esophagus, stomach (glandular and nonglandular), small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), pancreas, liver (at least 2 lobes), kidneys, urinary bladder, ovaries, uterus (plus cervix), mammary glands, testes, prostrate, seminal vesicles, skeletal muscle, skin and subcutis, eyes, Harderian glands, spinal cord, sciatic nerve, brain (cerebrum and cerebellum), and any other tissues with unusual lesions. The urinary bladder and lungs were inflated with fixative to facilitate their examination. Histopathological examinations and diagnoses were done by staff pathologists.


322

TAMANO ET AL

TABLE1.-Tumor incidences and survival rates of B6C3F, mice in different control groups. No. of mice Experiment

Effectiveb

Tumorbearing(%)

M 50 48 50 F' 50 50 47 2 M 48 F 50 3 M 50 50 F 50 49 4 M 50 49 49 F 50 50 5 M 50 F 50 50 250 244 Total M . F 250 246 M males; E females.

35 (72.9) 31 (62.0) 26 (55.3) 23(47.9) 38 (76.0) 24 (49.0) 36 (73.5) 32(65.3) 38 (76.0) 31 (62.0) 173 (70.9) 141 (57.3)

.

~

Sex# Initial

1

lymphomaslleukemias 34; others 18; glomerulonephritis 2; unknown 8). The numbers of benign and malignant tumors are shown in Table 11. In all, 243 tumors (1 13 benign and 130 malignant) were found in males, and 183 tumors (78 benign and 105 malignant) in females.

Survival rate in week 104

(96)

37 (74) 32 (64) 33 (66) 35 (70) 37 (74) 39 (78) 35 (70) 39 (78) 36 (72) 37 (74) 178 (7 1) 182 (73)

Orgaii Distribtitioii and Histological Classijication of Tiiinors The incidences of neoplasms are shown in Table 111.

~

0

*Total number of mice examined after the first tumor was found. RESULTS

Tumor Incidences and Siirvival Data The incidences of tumors and the survival rates of mice in each group are summarized in Table I. The incidences of tumors in all males and females in the 5 groups were 70.9% and 57.3%, respectively, the difference between the incidences in males and females being significant (p < 0.0 1). The incidence of tumors in the 5 groups varied slightly but not significantly from 55.3-76.0% in males and 47.965.3% in females. The first tumor was found in a male in week 79 (a hepatocellular carcinoma) and in a female in week 59 (an unclassified sarcoma of the uterus). The survival rates in the 5 experiments varied slightly, but not significantly, and so data on each sex in the 5 groups were combined. No differences between the survival rates of males and females was observed. Probable cause of death was ascertained for all animals which died spontaneously, which included 72 males (tumor associated: hepatocellular carcinomas 27; malignant lymphoma/ leukemias 13; others 15; inflammation associated: penis 2; other 1; unknown 14)and 68 females (tumor associated: hepatocellular carcinomas 6; malignant

TABLE 11.-Numbers of B6C3F, mice bearing sponta-

neous tumors.

Sex

Male Female

Total no. of mice

244 246

No.of mice with tumors

No.of tumors

(W

Total

Benign

173(70.9) 141 (57.3)

243 183

113

78

Malignant

130 105

TOXICOLOGIC PATHOLOGY

.

Circulatory Systein Hemangomas and hemangiosarcomaswere found in 6.1% and 2.5% of the males and 4.5% and 3.7% of the females, respectively. They developed in a variety of tissues including the liver (36.6%), uterus (19.5%), spleen (17.1%), skidsubcutis (4.9%), testis (4.9%), lymph nodes (2.4%), multiple organs (7.3%), and several other organs that had another primary lesion (7.3%). The hemangiosarcomas had irregular structures and the endothelial cells show marked atypic polymorphism. In 5 of 6 males and 2 of 9 females, the hemangiosarcomas metastasized to several tissues or organs, such as the lymph nodes, bone marrow, spleen, liver, pancreas, or lungs. Endocrine System The incidences of tumors of the endocrine system were low except for that of pituitary adenomas in females. Most of the pituitary tumors were benign and resembled chromophobic adenomas in appearance on H&E staining, although some seemed to be acidophilic adenomas. One pituitary tumor that had invaded the surrounding tissue in a female mouse was diagnosed as an adenocarcinoma. Thyroid adenomas were observed in 1 male and 2 females. They were composed of cuboidal cells that tended to fill the follicles and show microfollicular aspects. In the adrenal, a cortical adenoma was observed in 1 male and pheochromocytomas were found in 3 males and 1 female. One neuroblastoma originating from ganglion cells was found in the adrenal of a female. Islet-cell adenomas were rare, and were found incidentally on microscopical examination. Respiratory System Tumors of the upper respiratory tract were very rare. One adenocarcinoma of the nasal cavity was observed. Tumors of the lung were more frequent in males than in females. None of the 17 carcinomas metastasized. Digestive System Squamous cell papilloma developed at low incidence in the forestomach in both sexes and squa-


TABLE 111.-Incidences of primary tumors in control B6C3F, mice. No. of mice with tumor (%)

Site

Circulatory system Endocrine system Pituitary Thyroid Adrenal Pancreas Respiratory system Nasal cavity Lung Digestive system Tongue Esophagus Forestomach Large intestine Liver

Urinary system Kidney Reproductive system Testis Epididymis Ovary Uterus

Vagina Mammary gland Integumentary system

Special sense organs Harderian gland Body cavities Peritoneum Hematopoietic system Site unknown

Tumor type

Male

Female

Hemangioma Hemangiosarcoma

15 (6.1) 6 (2.5)

11 (4.5) 9 (3.7)

Adenoma Adenocarcinoma Adenoma Cortical adenoma Pheochromocytoma Neuroblastoma Islet-cell adenoma

l(0.4) 0 1 (0.4) l(0.4) 3 (1.2) 0 0

17 (6.9) 1 (0.4) 2 (0.8) 0 l(0.4) l(0.4) 3 (1.2)

Adenocarcinoma Adenoma Adenocarcinoma Squamous cell carcinoma Squamous cell carcinoma Squamous cell papilloma Squamous cell carcinoma Leiomyoma Leiomyosarcoma Hyperplastic nodule (HN) Hepatocellular carcinoma (HCC) Total HN and/or HCC Fibrosarcoma Unclassified sarcoma

0

22 (9.0) 12 (4.9) l(0.4) l(0.4) l(0.4) 0 0 0 64 (26.2) 58 (23.8) 117 (48.0) l(0.4) l(0.4)

l(0.4) 8 (3.3) 5 (2.0)

0 0 2 (0.8) 2 (0.8) l(0.4) l(0.4) 14 (5.7) 11 (4.5) 25 (10.2) 0 0

Transitional cell carcinoma

l(0.4)

0

Seminoma Leiomyosarcoma Adenoma Cyst adenoma Teratoma Endometrial stromal polyp Endometrial stromal sarcoma Leiomyosarcoma Unclassified sarcoma Adenocarcinoma Fibroadenoma Adendcarcinoma Sebaceous adenoma Fibroma Fibrosarcoma Malignant fibrous histiocytoma

l(0.4) l(0.4) NA NA NA NA NA NA NA NA 0 0 0 6 (2.5) 7 (2.9) 8 (3.3)

NA NA l(0.4) 3 (1.2) 1 (0.4) l(0.4) l(0.4) 5 (2.0) 2 (0.8) l(0.4) l(0.4) 3 (1.2) l(0.4) 1 (0.4) 2 (0.8) 3 (1.2)

Adenoma Adenocarcinoma

6 (2.5) l(0.4)

0

Lipoma Liposarcoma Malignant lymphomdleukemia Leiomyosarcoma Malimant fibrous histiocvtoma -

0 0

l(0.4) l(0.4)

NA: not applicable. Downloaded from tpx.sagepub.com by guest on July 10, 2011

35 (14.3) l(0.4) l(0.4) . .

7 (2.8)

58 (23.6)

0 0

TOXICOLOGIC PATHOLOGY

TAMANO ET AL

324

TABLE 1V.-Neoplasias occumng with more than 2.0% incidence in aging B6C3FI male mice. Percent risk

Site and tumor type Hematopoietic system Malignant lymphomdeukemia circulaiory system Hemangioma Hemangiosarcoma Lung Adenoma Adenocarcinoma Liver Hyperplastic nodule Hepatocellular carcinoma Integument Fibroma Fibrosarcoma Malignant fibrous histiocytoma Hardenan gland Adenoma No.at risk

No. of mice

Percent

35

14.3

15 6

6.1 2.5

22 12

9.0 4.9

64 58

26.2 23.8

6 7 8

2.5 2.9 3.3

6

2.5

244

mous cell carcinoma was found at low incidence in the tongue (male, 0.4%), esophagus (male, 0.4%), and forestomach (female, 0.8%). A high incidence of hepatocellular neoplasms was recorded. Hyperplastic nodules, characterized by compression of the surrounding parenchyma (equivalent to the hepatocellular adenomas of other authors, cf. 2, 9) and regarded asneoplastic lesions, were noted in 26.2% of the males and 5.7% of the females. The incidence of hepatocellular carcinomas was very similar to that of hyperplastic nodules in both sexes. Almost all the hepatocellular carcinomas were composed of hepatocytes, which frequently showed a prominent trabecular pattern diagnostic of well-differentiated carcinomas. Of the 5 8 hepatocellular carcinoma in male mice, 12 (20.7%) metastasized to the lung and 2 also to the kidney. Two (18.2%) of 11 carcinomas in females also metastasized to the lung. Other tumors of the digestive tract were leiomyoma and leiomyosarcoma of the large intestine in females. Urinary System Renal pelvic carcinoma, originating from transitional cells, was found in 1 male. No tumors of the urinary bladder were observed. Reprodiictise System In females, neoplasms were observed in several organs or tissues includingthe ovary, uterus, vagina,

0-52

weeks

53-78

weeks 8.3

8.3

16.7

79-104

weeks

105 weeks

28.6

11,2

8.2 6.1

6.2 1.7

8.2 8.2

9.6 4.5

16.3 46.9

31.5 18.5

8.2 6.1

3.4 1.7 2.2 3.4

5

12

49

178

and mammary gland, although their incidenceswere low. Leiomyosarcomas of the uterus (2.0%) were fairly common. In males, 1 seminoma in 1 testis and 1 leiomyosarcoma of the epididymis were observed.

Integurnentaiy System The incidence of fibrosarcomas originating in the skin were higher in males than females. These tumors were sometimes associated with skin lesions including inflammation in males, which fought periodically. Malignant tumors including fibrosarcomas and malignant fibrous histiocytomas were more frequent than benign fibromas and only 1 sebaceous adenoma was found. Special Sense Organs Harderian gland adenomas developed in 6 males (2.5%) and 7 females (2.8%); and an adenocarcinoma developed in 1 male. These tumors were mainly unilateral and were usually found in the final stage of the experiment. Body Cavities One lipoma and 1 liposarcoma were found in the peritoneal cavity of females but not males and no tumors developed in the pleural cavity.


Vol. 16,No. 3, 1988

SPONTANEOUS TUMOR IN B6C3F, MICE

325

TABLE V.-Neoplasias occumng with more than 2.0%incidence in aging B6C3F,female mice. Percent risk

Site and tumor type Hematopoietic system Malignant lymphomalleukemia CirculatoG system Hemangioma Hemangiosarcoma Pituitary Adenoma Lung Adenoma Adenocarcinoma Liver Hyperplastic nodule Hepatocellular carcinoma Uterus Leiomyosarcoma Harderian gland Adenoma No. at risk

weeks

53-78

79-104 weeks

105 weeks

23.6

20.0

66.0

11.5

11

9

4.5 3.7

10.0

3.8 13.2

4.9 0.5

17

6.9

1.9

8.8

8 5

3.3 2.0

5.7 1.9

2.7

14 11

5.7 4.5

1.9 11.3

7.1

5

. 2.0

1.9

2.2

7

2.8

NO.of mice

Percent

58

246

Heinatopoietic System Malignant lymphomasfleukemias were among the most common tumors, their incidence being higher in females (23.69'0) than in males (14.3%). Splenomegaly was commonly seen macroscopically. Enlargement of the liver and lymph nodes was also frequently apparent. The other tumors found were 1 leiomyosarcoma and 1 malignant 'fibrous histiocytoma of unknown origin in males. The common tumors with incidences of 2% or more are listed in Tables IV and V. Almost all the neoplasms developed more frequentlyin older mice, except hemangiosarcomas,fibrosarcomasofthe skin, and malignant fibrous histiocytomas in males, and hepatocellular carcinomas in females. DISCUSSION The incidence of tumor-bearing B6C3FI mice observed in this study was 70.9% in males and 57.3% in females. These values were similar to those reported by others (2, 9). The most common tumors in males were hyperplastic nodules of the liver, hepatocellular carcinomas, malignant lymphomas/ leukemias, adenomas and adenocarcinomas of the lung and hemangiomas. The most common tumors in females were malignant lymphomasAeukemias, pituitary adenomas, and hyperplastic nodules of the

0-52

weeks

20.0

2.2

1.6

3.8

1

10

53

182

liver. These results are also in general agreement with those reported previously (2, 5, 7, 9). But in the present study, the incidences of tumors in some organs were slightly different from those reported previously. For example, the incidences of tumors of the circulatory system, pituitary adenomas, hyperplastic nodules of the liver, hepatocellular carcinomas, fibrosarcomas of the skin, adenomas of the Harderian glands and malignant 1ymphomasAeukemiaswere higher than those reported by Ward et al (9). Moreover, the incidences of hemangiomas and hyperplastic nodules of the liver in males were higher than those reported by Haseman et a1 (2). These differenceswere probably attributable to different diagnoses of neoplasms in different laboratories (2, 5, 7). Moreover, the incidence ofspontaneous tumors depends on the genetic background, hormones, unknown environmental factors in the diet, air, water, or bedding and age (observation period) (2, 10). Some of these factors might cause slight differences in the observed incidences of spontaneous tumors. However, our survival rates compared favorably with those reported previously. The incidences of tumors of the endocrine system were low except for pituitary adenomas in female B6C3FI mice, possibly due to hormonal effects. Alveolarhronchiolar tumors are common in B6C3F,


326

TAMANO ET AL

mice, but are one of the neoplasms showing interlaboratory variability. The incidences of hepatic neoplasms were high due to genetic factors in this strain (3,6). In general, the extents of metastases of malignant neoplasms in laboratory animals vary (I), but hepatocellular carcinoma readily metastasized to the lung as reported by Vesselinovitch et al (8). Fibrosarcomas were sometimes associated with skin lesions including abscesses in males which fought periodically. Primary neoplasms of the Harderian glands are a peculiar characteristic of this species, and were frequent in both sexes. When grossly visible, they appeared as a round, raised area on the surface of the gland. Microscopically, papillary adenomas were the most frequent type. Papillary adenomas have been classified into 4 histological types: papillary, cystic papillary, acinar, and cystic by Sheldon et a1 (4).

In both sexes, the development of spontaneous tumors was age-related. The incidences of tumors increased from week 79 of the experiments in both sexes. Hepatocellular carcinomas, especially malignant lymphomas/leukemias in both sexes, fibrosarcomas in males and uterine tumors in females were sometimes the cause of death. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid from the Ministry of Health and Welfare of Japan and a Grant from the Society for Promotion of Pathology of Nagoya.

TOXICOLOGIC PATHOLOGY REFERENCES

1. Frith CH (1983). Incidence of hepatic metastases for various neoplasms in several strains of mice. Toxicol. Pathol. 11: 120-128. 2. Haseman JK, Huff J, and Boorman GA (1984). Use of historical control data in carcinogenicity studies in rodents. Toxicol. Pathol. 12: 126-153. 3. Heston WE and Vlahakis (1968). C3H-Av-A high hepatoma and high mammary tumor strain of mice. J. Natl. Cancer Inst. 40: 1161-1 166. 4. Sheldon WG, Curits M yKodell RL, and Weed L (1983). Primary Harderian gland neoplasms in mice. J. Natl. Cancer Inst. 71: 61-68. 5. Sher SPYJensen RD, and Bokelman DL (1982). Spontaneous tumors in control F344 and Charles RiverCD rats and Charles River CD-1 and B6C3F1 mice. Toxicol. Lett. 11: 103-1 10. 6. Stout DL and Becker FF (Tp86). Xenobiotic metabolizing enzymes in genetically and chemically initiated mouse liver tumors. CancerRes. 46: 2693-2696. 7. Tarone RE, Chu KC, and Ward JM (1981). Vanatility in the rates of some common naturally occumng tumors in Fischer 344 rats and (C57BU6N x C3WHeN)F1 (B6C3F1)mice. J. Natl. Cancer Inst. 66: 1175-1181. 8. Vesselinovitch SD, Mihailovich N, and Kao RK (1978). Morphology and metastatic nature of induced hepatic nodular lesions in C57BL x C3HF, mice. Cancer Res. 38: 2003-2010. 9. Ward JM, Goodman DG, Squire RA, Chu KC, and Linhart MS (1979). Neoplastic and nonneoplastic lesions in aging (C57BU6N x C3WHeN)Fl (136C3F1) mice. J. Natl. Cancer Inst. 63: 849-854. 10. Ward JM (1983). Background data and variations in tumor rates of control rats and mice. Prog. Exp. Tiiiiior Res. 26: 241-258,
