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Induction of adenocarcinomas in the submandibular salivary glands of female Wistar rats treated with 7,12-dimethylbenz(a)anthracene
Pergamon
Archs oral Biol. Vol. 41, No. 2, pp. 221-224, 1996 Copyright © 1996.ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969(95)00124-7 0003-9969/96$15.00+ 0.00
SHORT COMMUNICATION I N D U C T I O N OF A D E N O C A R C I N O M A S IN THE S U B M A N D I B U L A R SALIVARY GLANDS OF FEMALE WlSTAR RATS TREATED WITH 7,12-DIM ETHYLBENZ(A)ANTHRACENE A. ZAMAN, T. KOHGO, M. SHINDOH, T. IIZUKA and A, AMEMIYA Department of Oral Pathology, School of Dentistry, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo-060, Japan (Accepted 24 October 1995)
Sununary--In 12 male and 12 female Wistar rats, 7-9 weeks of age, a solution of 0.05ml of 1% 7,12-dimethylbenz(a)anthracene(DMBA) dissolved in acetone was injected directly into the submandibular glands biweekly, after the gland had been injured. In some rats the carcinogen was injected 1 week after the injm3,. Each rat received six to seven injections. Swelling was observed in the submandibular gland region as early as 4 weeks after the lastinjection. The animals were killed 4-8 weeks after the last injection and iglandswith tumour tissues were processed for light microscopy. The control rats of the same age that receipted a corresponding amount of acetone only (three male, three female),carcinogen injection alone (three male, five female), and injury only (four male, four female) were killed at the same time. Histological examination revealed adenocarcinomas of the submandibular gland in 12/12 (100%) female rats, six of which were associated with fibrosarcomas. The adenocarcinomas basically consisted of ductal and glandular structures. Sometimes tubular, cystic, papillary-cystic and cribriform-like structures were also observed. Male rats mainly developed fibrosarcomas, although there was one squamous-cell carcinoma. The reasons for the sex difference are not known. One adenocarcinoma developed in the submandibular gland of a female rat (1/5) that received carcinogen injections alone. Thus direct injections of DMBA into the submandibular gland produce adenocarcinomas in female Wistar rats. Key words: rat, female, submandibular gland, DMBA, adenocarcinoma.
Tumours in the salivary glands of rodents have generally been induced by local application of chemical carcinogens to the glandular structures using a variety of procedures. Squamous-cell carcinomas and fibrosarcomas are most commonly produced when chemicals are introduced into the salivary glands (Shafer, 1962; Chaudhry, Liposky and Jones, 1966; Schmutz and Chau,dhry, 1969; Standish and Shafer, 1960; Takai et al., 1984). There are a few reports of the induction of adenocarcinomas. Cataldo, Shklar and Chauncey (1964) induced adenocarcinomas in the submandibular glands of rats by local application of DMBA to the gland, and Cohen et al. (1975) produced many salivary adenocarcinomas in female Sprague-Dawley r~tts (male rats were not used) by feeding the animals 2-amino-4-(p-nitrophenyl) thiazole. We have now attempted to induce adenocarcinomas in the submandibular gland of rats by injecting the chemical carcinogen DMBA directly into the gland after injuring it. The purpose of the injury was to enhance tumour induction.
Twenty-four Wistar rats (12 male, 12 female), 7-9 weeks of age, were used as experimental animals. Under diethyl ether anaesthesia, a small incision was made on the ventral surface of the neck and both submandibular glands were exposed by blunt dissection. Then 0.05ml of 1% DMBA (Wako Pure Chemical Industries, Osaka, Japan) dissolved in acetone was injected into each gland after excision of a small portion of tissue (approx. 2 x 2 x 2 mm) from the body of the gland. In some animals (four male, five female) the carcinogen was injected 1 week after injury. The incision was closed carefully with silk thread. Equal amounts of the carcinogen were then injected biweekly, with each animal receiving six or seven injections. The control animals, male and female rats of the same age, were divided into three groups. Three males and three females were injected with a corresponding amount of acetone only, three males and five females with carcinogen alone, and in four males and four females the injury only was applied. The animals were separated according to sex, carefully monitored and regularly weighed throughAbbreviations: DMlq;A, 7,12-dimethylbenz(a)anthracene; out the entire experimental period. Experimental PAS, periodic acid-Schiff. animals, along with controls, were killed 4-8 weeks 221
222
A. Zaman et al.
Fig. 1. Dissection reveals a tumour with nodulation in the submandibular gland region in a female rat.
after the last DMBA injection by diethyl ether overdose. The submandibular glands with tumour tissue were removed. The tissue was placed in 10% neutralbuffered formalin for 48 h, processed in paraffin, sectioned at 4 p m and stained with haematoxylin and eosin and PAS for microscopic examination. In addition, lymph nodes and lungs were inspected and sections prepared to look for microscopic evidence of metastases. All male and female rats that received DMBA injections into the submandibular gland developed tumours in the neck region. In the females they were soft, encapsulated, nodular masses (Fig. 1) with a grey-white cut surface surrounded by soft fibrous tissue. Occasionally, cystic and necrotic areas were also noted. Tumours in the male rats were comparatively hard and encapsulated but nodulations were rarely seen. Histological examination revealed adenocarcinomas in the submandibular glands of the
female rats (12/12, 100%), six of which were associated with fibrosarcomas. Adenocarcinomas appeared as nodular masses and in most cases the carcinomas were adjacent to the glands. Basically, the adenocarcinomas consisted of glandular and ductal structures (Fig. 2). However, tubular, papillary-cystic, cystic and cribriform-like structures were occasionally seen in some animals (Fig. 3). The glandular and ductal structures were composed of cuboidal and low columnar cells that had large, pale-staining or hyperchromatic nuclei with prominent nucleoli. Mitoses and atypical configurations were noted in some areas. Local invasion into the surrounding tissue was detected but no metastases were evident in the lymph nodes and lungs. Male rats developed fibrosarcomas (11/12) and one squamous-cell carcinoma. The animals that received multiple carcinogen injections without prior injury developed one adenocarcinoma in the submandibular gland of a female rat. Other tumours noted in these animals were squamous-cell carcinomas (two females) and fibrosarcomas (two females, five males). No tumours were noted in the submandibular glands of the control animals that received acetone injections alone and injury only. We have induced adenocarcinomas in the submandibular glands of female rats by directly injecting DMBA into the gland; in contrast, fibrosarcomas were induced in most male rats. Cohen e t al. (1970, 1975) and Erturk, Cohen and Bryan (1970) induced adenocarcinomas in the salivary gland of female rats by feeding the animals 2-amino-4-(p-nitrophenyl)thiozole,2-hydrazino-4-(4-nitrophenyl)thiazole and 2-hydrazino-4-(4-aminophenyl)thiazole, or N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide. Heiman and Meisel (1946) also produced adenocarcinoma of the submandibular gland in female rats by feeding the
Fig. 2. A nodular growth of tumour in the submandibular gland in a female rat showing ductal, glandular and cyst-like structures, and stroma composed of scant fibrous connective tissue with small inflammatory elements. At the left-hand side there is intact salivary gland. Haematoxylin and eosin, x 40.
Adenocarcinoma in rat submandibular gland
223
Fig. 3. Adenocarcinoma in female rat showing papillary cystic pattern. PAS-positivesecretion can be seen in the cystic spaces. PAS, x 160.
animals 2-acetylaminofluorene. Both male and female rats were used in this study but adenocarcinomas developed only in female rats, whereas male rats developed sa:rcomas. It appears that adenocarcinomas are more commonly induced in female rat salivary glands. However, Steiner (1942), Bauer and Byrne (1950), Cataldo et al. (1964) and Takai et al. (1984) induced submandibular adenocarcinomas in both male and female rats and mice. The reasons for the sex differences are not clear. Cataldo and Shklar (1964) reported that fibrosarcomas rather than adenocarcinomas developed in the submandibular gland of hamsters, suggesting that there are species as well as sexual differences in outcome. Histologically, the adenocarcinomas consisted of ductal and glandular structures, and the tumours were invasive and spread into the surrounding fatty and connective tissue but metastases to the lymph nodes and lungs were not observed. These features have also been described by others (Cohen et al., 1975; Cataldo et ~l., 1964; Heimen and Meisel, 1946; Takai et al., 1984). Occasionally, tumours showed tubular, papillary--cystic and cribriform-like morphological features, some of which were also noted by Heimen and Meisel (1946). In man, adenocarcinoma is a rare tumour and comprises less than 10% of all salivary gland neoplasms (Batasakis, E1-Naggar and Luna, 1992). Histologically, solid, tubular, papillary and mucous morphological patterns have been described (Spiro, Huvas and Strong, 1982). Adenocarcinomas in the present study did not resemble the human counterpart but the different morphological patterns observed bore some resemblance to adenocarcinomas seen in man. It has been reported that the submandibular glands of rats regenerate after partial excision, mainly by forming actively proliferating duct-like structures
(Hanks and Chaudhry, 1971). We injured the gland to enhance the induction of tumours. After injury, the submandibular glands regenerated completely and no pathological lesions were clearly identified at the end of the experiment. It appears that injection of the carcinogen during the proliferative stage of healing in the submandibular gland, which followed 1 week after injury, and injection at the time of injury produce adenocarcinomas with almost equal frequency. However, the rate of development of adenocarcinomas was greater in rats that received DMBA injections with prior injury than in those without injury. This suggests that the injury may have been an influential factor in the development of adenocarcinomas. Further study is needed to clarify the roles of injury and sex hormones in carcinogenesis. We carefully examined other salivary glands, such as the parotid and sublingual, for evidence of the development of adenocarcinomas but no abnormal changes were detected. Tumours, in most cases, were identified adjacent to the submandibular gland. REFERENCES
Batsakis J. G., E1-Naggar A. K. and Luna M. A. (1992) "Adenocarcinoma, not otherwised specified": a diminishing group of salivary carcinomas. Ann. Otol. Rhinol. Laryngol. 101, 102-104. Bauer W. H. and Byrne J. J. (1950) Induced tumors of the parotid glands. Cancer Res. 10, 755-761. Cataldo E., Shklar G. and Chauncey H. (1964) Experimental submaxillary gland tumors in rats. Arch. Pathol. 77, 305-316. Cataldo E. and Shklar G. (1964) Chemical carcinogenesisin the hamsters submaxillary gland. J. dent. Res. 43, 568-579. Cohen S. M., Erturk E., Price J. M. and Bryan G. T. (1970) Comparative carcinogenicity in the rat of 2-hydrazinothiazoles with nitrofuryl, nitrophenyl, or aminophenyl substituents in the 4-position. Cancer Res. 30, 897-901.
224
A. Zaman et al.
Chaudhry A. P., Liposky R. and Jones J. (1966) Dose-response of submandibular glands to carcinogen pellets in rats and hamsters. J. dent. Res. 45, 1548-1550. Cohen S. M., Erturk E., Von Esch A. M., Crovetti A. J. and Bryan G. T. (1975) Carcinogenicity of 5-nitrofurans and related compounds with amino-hetero-cyclic substituents. J. natn. Cancer lnst. 54, 841-849. Erturk E., Cohen S. M. and Bryan G. T. (1970) Carcinogenicity of N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide in female rats. Cancer Res. 30, 936-941. Hanks C. T. and Chaudhry A. P. (1971) Regeneration of rat submandibular gland following partial extirpation. A light and electron microscopic study. Am. J. Anat. 130, 195-208. Heimen J. and Meisel D. (1946) Tumors of the salivary and parathyroid glands feed with 2-acetylaminofluorene. Cancer Res. 5, 617-619. Schmutz J. A. and Chaudhry A. P. (1969) Incidence of
induced tumors in the rat submandibular gland with different doses of 7,12-dimethylbenz(a)anthracene. J. dent. Res. 48, 1316. Shafer W. G. (1962) Experimental salivary gland tumorigenesis. J. dent. Res. 41, 117-124. Spiro R. H., Huvas A: G. and Strong E. W. (1982) Adenocarcinoma of salivary origin. Am. J. Surg. 144, 423-431. Standish S. M. and Shafer W. G. (1960) Experimental carcinogenesis in the duct-ligated and artery-ligated submaxillary salivary gland of the rat. Am. J. Pathol. 36, 473-478. Steiner P. E. (1942) Comparative pathology of induced tumors of the salivary glands. Arch. Pathol. 34, 613~524. Takai Y., Hyun K-H., Murase N., Hosaka M. and Mori M. (1984) Histopathologic studies of DMBA-induced mouse submandibular-gland tumors with or without cryoprobe treatment. J. oral Pathol. 13, 419-428.
Archs oral Biol. Vol. 41, No. 2, pp. 221-224, 1996 Copyright © 1996.ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969(95)00124-7 0003-9969/96$15.00+ 0.00
SHORT COMMUNICATION I N D U C T I O N OF A D E N O C A R C I N O M A S IN THE S U B M A N D I B U L A R SALIVARY GLANDS OF FEMALE WlSTAR RATS TREATED WITH 7,12-DIM ETHYLBENZ(A)ANTHRACENE A. ZAMAN, T. KOHGO, M. SHINDOH, T. IIZUKA and A, AMEMIYA Department of Oral Pathology, School of Dentistry, Hokkaido University, Kita-13, Nishi-7, Kita-ku, Sapporo-060, Japan (Accepted 24 October 1995)
Sununary--In 12 male and 12 female Wistar rats, 7-9 weeks of age, a solution of 0.05ml of 1% 7,12-dimethylbenz(a)anthracene(DMBA) dissolved in acetone was injected directly into the submandibular glands biweekly, after the gland had been injured. In some rats the carcinogen was injected 1 week after the injm3,. Each rat received six to seven injections. Swelling was observed in the submandibular gland region as early as 4 weeks after the lastinjection. The animals were killed 4-8 weeks after the last injection and iglandswith tumour tissues were processed for light microscopy. The control rats of the same age that receipted a corresponding amount of acetone only (three male, three female),carcinogen injection alone (three male, five female), and injury only (four male, four female) were killed at the same time. Histological examination revealed adenocarcinomas of the submandibular gland in 12/12 (100%) female rats, six of which were associated with fibrosarcomas. The adenocarcinomas basically consisted of ductal and glandular structures. Sometimes tubular, cystic, papillary-cystic and cribriform-like structures were also observed. Male rats mainly developed fibrosarcomas, although there was one squamous-cell carcinoma. The reasons for the sex difference are not known. One adenocarcinoma developed in the submandibular gland of a female rat (1/5) that received carcinogen injections alone. Thus direct injections of DMBA into the submandibular gland produce adenocarcinomas in female Wistar rats. Key words: rat, female, submandibular gland, DMBA, adenocarcinoma.
Tumours in the salivary glands of rodents have generally been induced by local application of chemical carcinogens to the glandular structures using a variety of procedures. Squamous-cell carcinomas and fibrosarcomas are most commonly produced when chemicals are introduced into the salivary glands (Shafer, 1962; Chaudhry, Liposky and Jones, 1966; Schmutz and Chau,dhry, 1969; Standish and Shafer, 1960; Takai et al., 1984). There are a few reports of the induction of adenocarcinomas. Cataldo, Shklar and Chauncey (1964) induced adenocarcinomas in the submandibular glands of rats by local application of DMBA to the gland, and Cohen et al. (1975) produced many salivary adenocarcinomas in female Sprague-Dawley r~tts (male rats were not used) by feeding the animals 2-amino-4-(p-nitrophenyl) thiazole. We have now attempted to induce adenocarcinomas in the submandibular gland of rats by injecting the chemical carcinogen DMBA directly into the gland after injuring it. The purpose of the injury was to enhance tumour induction.
Twenty-four Wistar rats (12 male, 12 female), 7-9 weeks of age, were used as experimental animals. Under diethyl ether anaesthesia, a small incision was made on the ventral surface of the neck and both submandibular glands were exposed by blunt dissection. Then 0.05ml of 1% DMBA (Wako Pure Chemical Industries, Osaka, Japan) dissolved in acetone was injected into each gland after excision of a small portion of tissue (approx. 2 x 2 x 2 mm) from the body of the gland. In some animals (four male, five female) the carcinogen was injected 1 week after injury. The incision was closed carefully with silk thread. Equal amounts of the carcinogen were then injected biweekly, with each animal receiving six or seven injections. The control animals, male and female rats of the same age, were divided into three groups. Three males and three females were injected with a corresponding amount of acetone only, three males and five females with carcinogen alone, and in four males and four females the injury only was applied. The animals were separated according to sex, carefully monitored and regularly weighed throughAbbreviations: DMlq;A, 7,12-dimethylbenz(a)anthracene; out the entire experimental period. Experimental PAS, periodic acid-Schiff. animals, along with controls, were killed 4-8 weeks 221
222
A. Zaman et al.
Fig. 1. Dissection reveals a tumour with nodulation in the submandibular gland region in a female rat.
after the last DMBA injection by diethyl ether overdose. The submandibular glands with tumour tissue were removed. The tissue was placed in 10% neutralbuffered formalin for 48 h, processed in paraffin, sectioned at 4 p m and stained with haematoxylin and eosin and PAS for microscopic examination. In addition, lymph nodes and lungs were inspected and sections prepared to look for microscopic evidence of metastases. All male and female rats that received DMBA injections into the submandibular gland developed tumours in the neck region. In the females they were soft, encapsulated, nodular masses (Fig. 1) with a grey-white cut surface surrounded by soft fibrous tissue. Occasionally, cystic and necrotic areas were also noted. Tumours in the male rats were comparatively hard and encapsulated but nodulations were rarely seen. Histological examination revealed adenocarcinomas in the submandibular glands of the
female rats (12/12, 100%), six of which were associated with fibrosarcomas. Adenocarcinomas appeared as nodular masses and in most cases the carcinomas were adjacent to the glands. Basically, the adenocarcinomas consisted of glandular and ductal structures (Fig. 2). However, tubular, papillary-cystic, cystic and cribriform-like structures were occasionally seen in some animals (Fig. 3). The glandular and ductal structures were composed of cuboidal and low columnar cells that had large, pale-staining or hyperchromatic nuclei with prominent nucleoli. Mitoses and atypical configurations were noted in some areas. Local invasion into the surrounding tissue was detected but no metastases were evident in the lymph nodes and lungs. Male rats developed fibrosarcomas (11/12) and one squamous-cell carcinoma. The animals that received multiple carcinogen injections without prior injury developed one adenocarcinoma in the submandibular gland of a female rat. Other tumours noted in these animals were squamous-cell carcinomas (two females) and fibrosarcomas (two females, five males). No tumours were noted in the submandibular glands of the control animals that received acetone injections alone and injury only. We have induced adenocarcinomas in the submandibular glands of female rats by directly injecting DMBA into the gland; in contrast, fibrosarcomas were induced in most male rats. Cohen e t al. (1970, 1975) and Erturk, Cohen and Bryan (1970) induced adenocarcinomas in the salivary gland of female rats by feeding the animals 2-amino-4-(p-nitrophenyl)thiozole,2-hydrazino-4-(4-nitrophenyl)thiazole and 2-hydrazino-4-(4-aminophenyl)thiazole, or N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide. Heiman and Meisel (1946) also produced adenocarcinoma of the submandibular gland in female rats by feeding the
Fig. 2. A nodular growth of tumour in the submandibular gland in a female rat showing ductal, glandular and cyst-like structures, and stroma composed of scant fibrous connective tissue with small inflammatory elements. At the left-hand side there is intact salivary gland. Haematoxylin and eosin, x 40.
Adenocarcinoma in rat submandibular gland
223
Fig. 3. Adenocarcinoma in female rat showing papillary cystic pattern. PAS-positivesecretion can be seen in the cystic spaces. PAS, x 160.
animals 2-acetylaminofluorene. Both male and female rats were used in this study but adenocarcinomas developed only in female rats, whereas male rats developed sa:rcomas. It appears that adenocarcinomas are more commonly induced in female rat salivary glands. However, Steiner (1942), Bauer and Byrne (1950), Cataldo et al. (1964) and Takai et al. (1984) induced submandibular adenocarcinomas in both male and female rats and mice. The reasons for the sex differences are not clear. Cataldo and Shklar (1964) reported that fibrosarcomas rather than adenocarcinomas developed in the submandibular gland of hamsters, suggesting that there are species as well as sexual differences in outcome. Histologically, the adenocarcinomas consisted of ductal and glandular structures, and the tumours were invasive and spread into the surrounding fatty and connective tissue but metastases to the lymph nodes and lungs were not observed. These features have also been described by others (Cohen et al., 1975; Cataldo et ~l., 1964; Heimen and Meisel, 1946; Takai et al., 1984). Occasionally, tumours showed tubular, papillary--cystic and cribriform-like morphological features, some of which were also noted by Heimen and Meisel (1946). In man, adenocarcinoma is a rare tumour and comprises less than 10% of all salivary gland neoplasms (Batasakis, E1-Naggar and Luna, 1992). Histologically, solid, tubular, papillary and mucous morphological patterns have been described (Spiro, Huvas and Strong, 1982). Adenocarcinomas in the present study did not resemble the human counterpart but the different morphological patterns observed bore some resemblance to adenocarcinomas seen in man. It has been reported that the submandibular glands of rats regenerate after partial excision, mainly by forming actively proliferating duct-like structures
(Hanks and Chaudhry, 1971). We injured the gland to enhance the induction of tumours. After injury, the submandibular glands regenerated completely and no pathological lesions were clearly identified at the end of the experiment. It appears that injection of the carcinogen during the proliferative stage of healing in the submandibular gland, which followed 1 week after injury, and injection at the time of injury produce adenocarcinomas with almost equal frequency. However, the rate of development of adenocarcinomas was greater in rats that received DMBA injections with prior injury than in those without injury. This suggests that the injury may have been an influential factor in the development of adenocarcinomas. Further study is needed to clarify the roles of injury and sex hormones in carcinogenesis. We carefully examined other salivary glands, such as the parotid and sublingual, for evidence of the development of adenocarcinomas but no abnormal changes were detected. Tumours, in most cases, were identified adjacent to the submandibular gland. REFERENCES
Batsakis J. G., E1-Naggar A. K. and Luna M. A. (1992) "Adenocarcinoma, not otherwised specified": a diminishing group of salivary carcinomas. Ann. Otol. Rhinol. Laryngol. 101, 102-104. Bauer W. H. and Byrne J. J. (1950) Induced tumors of the parotid glands. Cancer Res. 10, 755-761. Cataldo E., Shklar G. and Chauncey H. (1964) Experimental submaxillary gland tumors in rats. Arch. Pathol. 77, 305-316. Cataldo E. and Shklar G. (1964) Chemical carcinogenesisin the hamsters submaxillary gland. J. dent. Res. 43, 568-579. Cohen S. M., Erturk E., Price J. M. and Bryan G. T. (1970) Comparative carcinogenicity in the rat of 2-hydrazinothiazoles with nitrofuryl, nitrophenyl, or aminophenyl substituents in the 4-position. Cancer Res. 30, 897-901.
224
A. Zaman et al.
Chaudhry A. P., Liposky R. and Jones J. (1966) Dose-response of submandibular glands to carcinogen pellets in rats and hamsters. J. dent. Res. 45, 1548-1550. Cohen S. M., Erturk E., Von Esch A. M., Crovetti A. J. and Bryan G. T. (1975) Carcinogenicity of 5-nitrofurans and related compounds with amino-hetero-cyclic substituents. J. natn. Cancer lnst. 54, 841-849. Erturk E., Cohen S. M. and Bryan G. T. (1970) Carcinogenicity of N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide in female rats. Cancer Res. 30, 936-941. Hanks C. T. and Chaudhry A. P. (1971) Regeneration of rat submandibular gland following partial extirpation. A light and electron microscopic study. Am. J. Anat. 130, 195-208. Heimen J. and Meisel D. (1946) Tumors of the salivary and parathyroid glands feed with 2-acetylaminofluorene. Cancer Res. 5, 617-619. Schmutz J. A. and Chaudhry A. P. (1969) Incidence of
induced tumors in the rat submandibular gland with different doses of 7,12-dimethylbenz(a)anthracene. J. dent. Res. 48, 1316. Shafer W. G. (1962) Experimental salivary gland tumorigenesis. J. dent. Res. 41, 117-124. Spiro R. H., Huvas A: G. and Strong E. W. (1982) Adenocarcinoma of salivary origin. Am. J. Surg. 144, 423-431. Standish S. M. and Shafer W. G. (1960) Experimental carcinogenesis in the duct-ligated and artery-ligated submaxillary salivary gland of the rat. Am. J. Pathol. 36, 473-478. Steiner P. E. (1942) Comparative pathology of induced tumors of the salivary glands. Arch. Pathol. 34, 613~524. Takai Y., Hyun K-H., Murase N., Hosaka M. and Mori M. (1984) Histopathologic studies of DMBA-induced mouse submandibular-gland tumors with or without cryoprobe treatment. J. oral Pathol. 13, 419-428.