Pulmonary fibrosis caused by N-methyl-N-nitrosourethane inhibits lung tumorigenesis by urethane in transgenic mice carrying the human prototype c-Ha-ras gene.

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.

Dose-response carcinogenicity in rats on low-dose levels of N-ethyl-N-nitrosourethane.

A dose-response study on the carcinogenicity of N-ethyl-N-nitrosourethane (ENUR) was undertaken to examine its effect at low doses. Six-week-old female F344 rats were divided into 5 groups, each consisting of 40 animals. ENUR was dissolved in distilled water at dose levels of 0 (control), 0.15, 0.6, 2.5 and 10 ppm, and rats were given these solutions ad libitum for 2 years. Significant increase of the total tumor incidences and shortening of the mean survival times were observed in groups given 2.5 and 10 ppm ENUR. In groups given 0.6 ppm or more ENUR, digestive tract tumors were induced dose-dependently. They were restricted to the upper digestive tract from the oral cavity to the forestomach, and were histologically squamous cell papillomas or carcinomas. Dose-related differences in the location and incidence of these tumors were found. The virtually safe doses (VSDs) calculated by using the Weibull, Logit and Probit models were 0.365 x 10(-2), 0.110 x 10(-1) and 0.779 x 10(-1) ppm, respectively. The VSDs estimated in the present study are discussed in comparison with those of other carcinogens.

Effect of exogenous surfactant instillation on experimental acute lung injury.

Pulmonary surfactant replacement has previously been shown to be effective in the human neonatal respiratory distress syndrome. The value of surfactant replacement in models of acute lung injury other than quantitative surfactant deficiency states is, however, uncertain. In this study an acute lung injury model using rats with chronic indwelling arterial catheters, injured with N-nitroso-N-methylurethane (NNNMU), has been developed. The NNNMU injury was found to produce hypoxia, increased mortality, an alveolitis, and alterations in the pulmonary surfactant system. Alterations of surfactant obtained by bronchoalveolar lavage included a reduction in the phospholipid-to-protein ratio, reduced surface activity, and alterations in the relative percentages of the individual phospholipids compared with controls. Treatment of the NNNMU-injured rats with instilled exogenous surfactant (Survanta) improved oxygenation; reduced mortality to control values; and returned the surfactant phospholipid-to-protein ratio, surface activity, and, with the exception of phosphatidylglycerol, the relative percentages of individual surfactant phospholipids to control values.

Effects of minute amounts of cigarette smoke with or without nebulized N-nitroso-N-methylurethane on the respiratory tract of mice.

The effects of minute amounts of cigarette smoke with or without nebulized N-nitroso-N-methylurethane (NMUT) on the respiratory epithelia of mice were examined. The first group received the combination of NMUT nebulization and cigarette smoke inhalation, the second group NMUT nebulization only, the third group cigarette smoke inhalation only and the fourth group no treatment. The first group showed squamous cell carcinomas in the nasal cavity (4 out of 10 mice), trachea (4 out of 10 mice) and lung (2 out of 10 mice). In the second group squamous cell carcinomas with keratosis were found in the nasal cavity (2 out of 10 mice) and in the lung (1 out of 10 mice). Furthermore, basal cell hyperplasias, squamous metaplasias and dysplastic changes were found in these two groups. The third group showed basal cell hyperplasias and squamous metaplasias in the nasal cavity and hypertrophy of Clara cells. No marked changes of epithelia of the respiratory tract in the fourth group were seen. Adenomas and adenocarcinomas were not found in any group. Statistically, there is a significant difference in the incidence of dysplastic change of the trachea between group 1 and group 2 (P less than 0.05). These results suggest that minute amounts of cigarette smoke, in combination with carcinogens, may have cocarcinogenic action and that squamous cell carcinoma may pass through the course of basal cell hyperplasia, squamous metaplasia and dysplastic change of the epithelia.

Carcinogenic effect of N-ethyl- and N-amyl-N-nitrosourethans on female Donryu rats.

Carcinogenic effect of N-ethyl-N-nitrosourethan (ENUR) and N-amyl-N-nitrosourethan (ANUR) was examined by continuous oral administration or topical application to female Donruy rats. Oral administration of 100 ppm solution of ENUR induced 100% of tumors in the forestomach, 46%, 80%, 71%, and 51% in the oral cavity and pharynx, esophagus, duodenum, and liver, respectively. On the other hand, the incidence of forestomach tumors was 78%, that of oral cavity and pharynx, and esophagus was 93% and 98%, respectively, in rats given 400 ppm suspension of ANUR. In addition, topical application of ENUR induced tumors of the skin as well as tumors of the forestomach and liver.

Alveolar epithelial injury causing respiratory distress in dogs: physiologic and electron-microscopic correlations.

Structural and mechanical changes were correlated in 29 dogs with acute alveolar injury induced by the subcutaneous administration of N-nitroso-N-methylurethane (NNNMU). The injury was characterized by necrosis and repair of alveolar epithelium while the vascular endothelium remained essentially intact. Compliance of the lung (CL) decreased and elastic recoil increased as epithelial necrosis occurred. During recovery, improved elastic recoil coincided with epithelial regeneration, although CL remained abnormal. The late phase was characterized histologically by widespread closure of clusters of alveoli alternating with dilated small air spaces. The process resulted in distortion of lung architecture and resembled interstitial fibrosis. Reduced lung volume and decreased distensibility of dilated small air spaces may have accounted for the persistently abnormal CL. Because of the specific site of anatomic involvement, the predictable evolution of deranged lung mechanics, and the similarity to human lung injury (adult respiratory distress syndrome), the lung injured by administration of N-nitroso-N-methylurethane is a suitable model to study pathophysiology and types of therapy in a controlled setting.

Histopathological studies on experimentally induced pulmonary, pleural and peritoneal neoplasms in mice by intraperitoneal injection of chrysotile asbestos and N-methyl-N-nitrosourethane.

The cocarcinogenic effects of asbestos are presented. In lung carcinomas induced in mice, the number of carcinomas and the time of detection of the first carcinoma per tumor-bearing animals were greater and faster in the group with chrysotile plus MNU than either chrysotile or MNU alone. This suggested that chrysotile asbestos had a promoting or cocarcinogenic effect on some carcinogens in the respiratory tract. In the group treated with chryotile alone, a tumor was found in the right pleural cavity at 15 months. This tumor microscopically was similar to the biphasic form of the human diffuse mesothelioma. Microvilli, basement membrane, and junctional apparatus were seen by electron microscope, but other cytoplasmic organelles of the tumor cells were relatively scanty. Two peritoneal tumors developed in gastric and intestinal serosa at 11 and 12 months. Light and electron microscopic studies suggested that the tumors were probably myosarcomas or fibrosarcoms.

Carcinogenic effect of N-butyl-N-nitrosourethan on CDF1 mice.

Studies were made on the induction of tumors in mice by N-butyl-N-nitro-sourethan (BNUR), with the following results. 1) Oral administration of 0.04% BNUR in deionized water to CDF1 mice for 20 weeks produced papillomas in the esophagus and forestomach of all 58 mice, and 24 of the mice had squamous cell carcinoma. 2) No skin tumors developed when 0.1%, 0.5%, or 1% BNUR in acetone was painted on the skin on the back of CDF1 mice three times a week for 25 weeks. 3) No sarcomas developed within 30 weeks when a dose of 10, 60, or 120 mg/ml of BNUR in dimethyl sulfoxide solution was injected subcutaneously into the back of CDF1 mice, 10 times at 1-week intervals. 4) In the in vitro malignant transformation test, golden hamster lung fibroblast cells did not show malignant transformation during an observation period of 150 days after treatment with BNUR at a final concentration of 30 and 60 mug/ml for 24 hr. The carcinogenicity and possible mode of action of BNUR are discussed on the basis of these results.

Histological changes in the submandibular glands of rats after intraductal injection of chemical carcinogens.

Carcinogens injected into the excretory canal of submandibular gland of Donryu rats revealed the following histologic changes in salivary glands. 20-Methylcholanthrene induced squamous cell metaplasia, fibrosis in the early stages, and "benign lymphoepithelial lesion"-like pattern after 3 months. Dense hyalinization occurred after 4-5 months with so-called "mixed tumor"-like pattern. In the later stages epidermoid carcinoma and fibrosarcoma were observed. 9, 10-Dimethylbenzanthracene caused degenerative change, metaplasia, fibrosis and cell infiltration, and later carcinoma and sarcoma appeared at a high rate. 4-Nitroquinoline-N-oxide led to dense hyalinization and so-called "mixed tumor"-like pattern was observed in many specimens. N-nitroso-N-methyl urethane and N-methyl-N-nitroso-N'-nitroguanidine revealed metaplastic changes, fibrosis and lymphoid infiltration. Scarlet red induced remarkable infiltration and aggregation of lymphoid cells, showing benign "lymphoepithelial lesion"-like pattern.