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To investigate mechanisms underlying accelerated carcinogenesis in mice carrying a human prototype c-Ha-ras gene (rasH2 mouse), mutations and the expression profile of the transgene were evaluated in 14 tumors induced by a single injection of ethylnitrosourea (ENU), with or without additional beta-estradiol 3-benzoate (EB) treatment. Although no codon 12 mutations were detected, changes in codon 61 were evident in all lung adenocarcinomas, skin squamous cell carcinomas and forestomach squamous cell carcinomas examined. The mRNA levels of the transgene in these lesions were also elevated 1.71- to 4.77-fold, 3.04- to 5.18-fold, and 3.00- to 5.67-fold, respectively, in comparison with those in the normal livers of rasH2 mice. The results obtained in this study suggest that mutations in codon 61 and amplification of the transgene play key roles in the carcinogenesis induced by ENU in rasH2 mice.

Previous studies showed that significant differences in mutation frequency of the human c-Ha-ras transgene between vinyl carbamate (VC)- and ethyl carbamate (urethane)-induced lung tumors were observed in rasH2 mice. It remains unclear why the point mutation frequency is extremely low in VC-induced lung tumors, although this compound is much more carcinogenic than urethane. In this study, we examined the somatic point mutations of the transgene at the RNA level in VC- and urethane-induced lung tumors of rasH2 mice. We did not find any mutation at codon 12 of the transgene in any of these lung tumors, but codon 61 showed frequent mutations in not only urethane-induced lung tumors (15 out of 16) but also VC-induced lung tumors (11 out of 11) in rasH2 mice. These results suggested that point mutations at codon 61 of the transgene play an important role in the carcinogenesis of VC- and urethane- induced lung tumors in rasH2 mice.

Previously we reported a lack of modification by cinnamaldehyde (CNMA) of development of lung proliferative lesions induced by urethane in CB6F1-TgHras2 (rasH2) mice. In the present study, we re-evaluated CNMA effects using the same rasH2 strain and non-transgenic littermates initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Sixteen mice/strain/sex received intraperitoneal NNK injections at a dose of 3 mg/mouse once a week for 2 weeks followed by free feeding of commercial diet containing 5000 ppm CNMA for 26 weeks. Additional groups were maintained without NNK injection and/or CNMA feeding for 28 weeks. Lung tumors were induced by NNK in both rasH2 and non-Tg males and females at incidence ranging from 63 to 100%. CNMA treatment significantly reduced the combined incidence of adenomas and carcinomas from 86 to 31% in rasH2 males (P<0.05), but no significant influence was evident in females. The multiplicity of NNK-induced lung tumors was also significantly reduced in rasH2 males given CNMA (P<0.01). Similar effects were also observed in non-Tg females given CNMA after NNK initiation. The results of our study strongly indicate that CNMA is capable of inhibiting development of NNK-initiated pulmonary tumorigenesis in rasH2 and non-Tg mice.

To investigate the modifying effects of eugenol (EUG), a component of cigarette smoke, on lung carcinogenesis, male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) were given a single intraperitoneal injection of 250 mg/kg urethane (UR) or saline, followed by a diet containing 6,000 ppm EUG or basal diet for 26 weeks. Their non-transgenic CB6F1 littermates (non-Tg mice) were also treated in the same manner. In both male and female rasH2 mice, alveolar/bronchiolar hyperplasias, adenomas and carcinomas were observed in all UR-treated groups. However, there were no significant intergroup differences in the incidences and multiplicities of these lesions between the UR alone and UR + EUG groups. In non-Tg mice, alveolar/bronchiolar hyperplasias, adenomas or carcinomas were sporadically observed in UR-treated groups of both sexes, with no significant differences in the incidences and multiplicities between the UR alone and UR + EUG groups. There were no intergroup differences between them in the PCNA-positive ratios of adenomas or carcinomas and the areas of adenomas or carcinomas to the whole lung area examined. The present results suggest that the EUG treatment does not exert modifying effects on lung carcinogenesis induced by UR in both male and female rasH2 mice and non-Tg mice.

In our previous study, when rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1,000 mg/kg of urethane once or three times at two-day intervals, the incidence of lung proliferative lesions in rasH2 mice given triple doses of urethane was significantly increased, compared to that in rasH2 mice given a single dose of urethane, and the mutation frequency of the transgene in lung tumors in rasH2 mice given triple doses was lower than that in rasH2 mice given a single dose of urethane. In the present study, differential immunohistochemical expressions of Cyclin D1 and PCNA, that lead to abnormal cell proliferation and tumor development due to uncontrolled G1-S transition in the cell cycle, as well as p53 tumor suppressor gene in pulmonary proliferative lesions obtained from our previous study were investigated. Over-expression of Cyclin D1 in hyperplasias in rasH2 mice given triple doses was significantly increased, compared to that in the single-injection group, but no significant differences in Cyclin D1 between the single and triple injection groups were observed in hyperplasias in non-Tg mice or lung tumors in either rasH2 or non-Tg mice. There were no differences in the PCNA labeling index of hyperplasias in rasH2 or non-Tg mice between the triple-injection and single-injection groups, while the PCNA labeling index tended to be increased in the tumor, compared with that in hyperplasias. There was neither mutation of p53 nor an increase in immunoreactivity of wild type p53 in these proliferative lesions. These results suggest that cyclin D1 over-expression in alveolar/bronchiolar hyperplasias in rasH2 mice in the triple-injection group is not only indicative of a high cell proliferation rate but also of an important role in the process of malignant transformation.

Urethane-induced lung tumors and their genetic changes were investigated in transgenic (Tg) mice carrying a human prototype c-Ha-ras gene (rasH2 mice). Male and female rasH2 mice and non-transgenic (non-Tg) littermates were injected intraperitoneally with 1000 mg/kg of urethane once or three times at 2-day intervals. Hyperplasias and adenomas of the lung were observed in all animals of each group from week 10, and carcinomas were observed in male and female rasH2 mice of the triple injection group from week 10 and female non-Tg mice of the single injection group at 15/20 weeks. The multiplicities of lung proliferative lesions including hyperplasias, adenomas and carcinomas, in treated rasH2 mice were significantly higher than those in treated non-Tg mice. CAG to CTG transversions were observed in the c-Ha-ras gene in these lung proliferative lesions of rasH2 mice of the single injection group at high incidence (male: 58.3%, female: 62.5%), but no mutations of the mouse c-Ki-ras gene were evident in either rasH2 or non-Tg mice. In the triple injection group, transgene mutations were detected at a relatively low incidence, and mouse c-Ki-ras gene mutations(CAA to CGA) were observed in both rasH2 and non-Tg mice. These results suggest that the variation of the lesions induced by different doses of urethane was not the cause of the variation of the mutation spectrum and mutations of both transgene and mouse c-K-ras gene are not principal genetic events in urethane-induced lung proliferative lesions in rasH2 mice.

Male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and their wild littermates (non-Tg mice) received three subcutaneous injections of 0.3 mg N-methyl-N-nitrosourethane (MNUR) once every 2 weeks for the first 4 weeks followed by a single intraperitoneal injection of 1000 or 0 mg/kg urethane (UR) 2 weeks later. They were then maintained without any other treatment for a further 13 weeks and sacrificed for assessment of pulmonary pathology. Inflammatory lesions, such as macrophage infiltration, alveolar bronchiolization and/or fibrosis, were induced in both rasH2 and non-Tg mice treated with MNUR or MNUR + UR. Lung proliferative lesions were induced in 100% of the UR-treated rasH2 mice but to a significantly lesser extent in the MNUR + UR case. The incidences of lung tumors in non-Tg mice treated with UR or MNUR + UR were relatively low. Point mutations of the transgene were detected in approximately 80% of lung tumors in rasH2 mice treated with UR and MNUR + UR, but murine Ki-ras mutations were rare. No marked difference in the mutation pattern was found between the UR-treated and the MNUR + UR-treated rasH2 mice. In non-Tg mice treated with UR or MNUR + UR, point mutations of the murine c-Ki-ras gene were observed in about 50% of the lung tumors examined. The present study confirmed that rasH2 mice are very sensitive to lung tumor induction by UR and suggested that alveolar epithelial cells in the reparative stage during pulmonary fibrosis are resistant to DNA damage by this carcinogen.

To determine if hemizygous transgenic mice carrying the human c-Ha-ras gene (CB6F1-Tg Hras2 mice (Hras2 mice)) are susceptible to the carcinogenic potential of known murine carcinogens, male and female Hras2 mice and their non-transgenic CB6F1 littermates (non-Tg mice) were each given a single intraperitoneal injection of 60 mg of vinyl carbamate (VC)/kg body weight or saline (vehicle control) and monitored for 16 wk without further treatment. At necropsy, grossly visible tumors were fixed for histopathologic diagnosis and, when of sufficient size, portions were frozen for subsequent molecular analysis. Nine of 31 male and nine of 29 female Hras2 mice treated with VC died within 16 wk as a result of lung tumor burden. At the termination of the study, lung tumors (alveolar-bronchiolar epithelial neoplasms and hemangiosarcomas) and focal alveolar-bronchiolar hyperplasias were present in both sexes of Hras2 and non-Tg mice treated with VC; there were significantly more proliferative lung lesions in Hras2 than non-Tg mice. Splenic hemangiosarcomas and squamous cell tumors of the forestomach were induced in male and female VC-treated Hras2 mice but not in VC-treated non-Tg mice. Polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing of the induced lung tumors revealed point mutations at codon 61 of the transgene in two of 29 lung tumors (one of 16 in males and one of 13 in females) from VC-treated Hras2 mice; no mutations in murine Ki-ras were found in these tumors. Point mutations at codons 12 and 61 of the murine Ki-ras gene were observed, however, in one of 10 and six of 10 lung tumors respectively, from VC-treated non-Tg mice. These findings indicate that Hras2 mice are highly sensitive to pulmonary neoplasms and splenic and lung hemangiosarcomas after treatment with VC. The molecular analyses suggest that point mutations of the transgene and the murine Ki-ras gene do not play a major role in VC induction of pulmonary neoplasms in these transgenic mice.

To establish the characteristics of an optimized pulmonary fibrosis model, male ICR mice were given 4 weekly sc injections of 150 or 0 mg/kg monocrotaline (MCT) and maintained without further treatment for 33 wk (Experiment 1). The final mortality in the MCT group was 64%. Epithelial cells with large bizarre nuclei and an increased incidence of alveolar/bronchiolar hyperplasias were typically observed. In areas of pulmonary fibrosis, the PCNA labeling index (LI) in the alveolar/airway epithelium was significantly elevated. DNA content analysis demonstrated a larger range (4-8C) for the ploidy pattern of alveolar epithelium with large bizarre nuclei than in the normal epithelium (2C). In Experiment 2, the relationship between pulmonary fibrosis development and lung tumorigenesis was investigated. Mice were given 4 weekly sc injections of 150 and 0 mg/kg MCT, followed by a single i.p. injection of 1,000 or 500 mg/kg urethane (UR) on week 7, then maintained without further treatment for an additional 15 wk. UR following MCT-induced inflammatory changes, fibrosis, and epithelia with large bizarre nuclei but no tumorous lesions, in spite of the fact that treatment with UR alone caused a high incidence of pulmonary tumors. Hyperplasias were seen in all groups, but the multiplicity in the combined groups tended to be decreased by the MCT pretreatment. The present study demonstrated that this new protocol is more suitable than previous one for the experimental production of pulmonary fibrosis. Furthermore, the induction of lung tumors by UR was completely depressed in mice with MCT-induced pulmonary fibrosis, suggesting that alveolar epithelial cells are resistant to this lung carcinogen under these conditions.

In order to investigate whether goitrogens and liver enzyme-inducers modify the tumorigenesis in the liver or lung, 6-week old male F344 rats were given single subcutaneous injection of DHPN, and starting one week later received water containing goitrogens, namely sulfadimethoxine (SDM), propylthiouracil (PTU) and potassium thiocyanate (KSCN), or an enzyme-inducer, phenobarbital (PB), for 19 weeks ad libitum. Although the number of GST-P positive foci in the liver was significantly increased in the PB group as compared to the control group, there were no significant fluctuations in the SDM, PTU and PB groups. With respect to the lung, it is suggested that SDM, KSCN and PB may enhance the lung tumorigenesis, since the multiplicities of hyperplasias of alveolar epithelia were increased in groups treated with these compounds.

To cast light on the relationship between the development of pulmonary fibrosis and lung cancer, female Syrian golden hamsters were given 5 sc injections of 0.6 mg/animal of N-methyl-N-nitrosourethane (MNUR) at 2-wk intervals and then maintained without any treatment for 26 wk. Bronchiolo-alveolar cell tumors and hyperplasias, which were recognized from week 4 after termination of treatment, were each subdivided morphologically into 3 types. Bronchiolo-alveolar cell tumors included papillary tumors consisting of basophilic cells, papillary tumors consisting of clear cells, and papillary/solid tumors consisting of pleomorphic cells, with papillary tumors consisting of basophilic cells predominating. Bronchiolo-alveolar cell hyperplasias encountered were glandular metaplasia, simple hyperplasia, and papillary hyperplasia. Glandular metaplasia was the most common of the hyperplastic lesions. To some extent, all the proliferative lesions were associated with inflammatory cell infiltration, but this varied greatly. The rate for papillary tumors consisting of basophilic cells with connective tissue proliferation was 35%. Among the hyperplastic lesions, the cell proliferative activity of papillary hyperplasia (12.5%) was significantly higher than in other hyperplastic lesions, suggesting that this lesion might be a preneoplastic change. None of the lung proliferative lesions showed any unequivocal immunoreactivity for the p53 protein. The present study suggests that most lung tumors may develop from pulmonary inflammatory lesions induced by MNUR, but the possibility that DNA injuries to the respiratory epithelial cells by MNUR may cause lung tumors cannot be precluded.