Frequent mutations of Ki-ras but no mutations of Ha-ras and p53 in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats

Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single-strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. © 1996 Wiley-Liss, Inc.     

Prostatic cancer induced in MRC rats by N-nitrosobis(2-oxopropyl)-amine and N-nitrosobis(2-hydroxypropyl)amine

Weekly intragastric treatment with N-nitrosobis(2-oxo-propyl)amineor N-nitrosobis(2-hydroxypropyl)amine induced hyperplastic,preneoplastic and neoplastic prostatic changes in >80% ofMRC rats. The lesions initially appeared as focal or multifocalproliferations of alveolar epithelium in a cribriform patternwhich, in all but one case, underwent progressive changes, oftentending toward squamous cell formation. Tumors, found primarilyin the ventral prostate, demonstrated various degrees of differentiationand invasive growth. A few neoplasms developed in the seminalvesicles; however all were of a glandular type. The sequentialalteration of induced lesions is described and the possiblereasons for the squamous cell character of most tumors discussed.Prostatic cancer induction by systemic application of specificnitrosamines could provide a unique tool for investigating importantaspects of the disease.     

Elevated expression of interleukins in lung adenocarcinomas induced by N-Nitrosobis(2-hydroxypropyl)amine in rats.

The expression of interleukins (ILs) in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats was investigated using a multiprobe RNase protection assay (RPA) followed by densitometric quantification. Male Wistar rats, 6 weeks old, were given 2000 ppm BHP in their drinking water for 12 weeks and maintained without further treatment until they were killed at week 25. Total RNAs were extracted from 14 individual adenocarcinomas and 2 specimens of normal lung tissue of untreated rats. In adenocarcinomas, elevated expression of IL-1alpha (6 / 14), IL-1beta (14 / 14), IL-3 (7 / 14), IL-4 (11 / 14), IL-5 (9 / 14), IL-6 (11 / 14) and IL-10 (8 / 14) was observed, compared with normal lung tissues. In contrast, no expression of IL-2 was detected in any case. The results suggest that preferential expression of these ILs and their complex networks may contribute to the development and progression of lung adenocarcinomas induced by BHP in rats.     

Increased expression of cyclooxygenase-2 protein in rat lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine

Expression of cyclooxygenase (COX)-2 protein in preneoplastic and neoplastic lung lesions induced by the administration of 2000 ppm of N-nitrosobis(2-hydroxypropyl)amine (BHP) in the drinking water to Wistar male rats, was examined immunohistochemically. The majority of alveolar/bronchiolar adenomas (ADs) and all adenocarcinomas (ADCs) examined, stained positive or strongly positive for COX-2. In contrast, only a minority of alveolar/bronchiolar hyperplasias demonstrated immunoreactivity and half of the squamous cell carcinomas examined, were only weakly positive. Western blotting analysis also revealed expression of COX-2 protein in the resected ADs and ADCs. These results clearly indicate up-regulated expression of COX-2 in lung neoplastic lesions, particularly ADs and ADCs, induced by BHP in rats.     

Carcinogenic potency of N-nitrosomethyl(2-hydroxypropyl)amine and other metabolic relatives of N-nitrosobis(2-hydroxypropyl)amine by single intraperitoneal injection on the lung of rats

The carcinogenic effects of a single intraperitoneal injection of N-nitrosobis(2-hydroxypropyl)amine (BHP) or its metabolic relatives, N-nitrosomethyl(2-hydroxypropyl)amine (MHP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-2,6-dimethylmorpholine (NDMM), were studied in male Wistar rats. The main target organ of these nitrosamines proved to be the lung, followed by the thyroid. Lung lesions were induced in a dose-dependent manner with total lung tumor incidences reaching 55% to 100%. BHP, MHP, HPOP and NDMM all caused lung carcinomas to develop (22% to 44% incidence), whereas BOP was only associated with adenomas. On the basis of dose administered and incidence of carcinomas, MHP appeared to be the most potent lung carcinogen of the five nitrosamines investigated. Smaller numbers of neoplasms were also induced in the kidney, urinary bladder, esophagus and intestine at differing rates by these nitrosamines.     

Mutations and reduced expression of the transforming growth factor-beta receptor II gene in rat lung adenocarcinomas induced by N-nitrosobis-(2-hydroxypropyl)amine.

Mutations and expression of the transforming growth factor-beta receptor type II (TGF-beta RII) gene were investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Males of the Wistar strain, 6 weeks old, were given 2000 ppm of BHP in their drinking water for 12 weeks and then maintained without further treatment until killed at week 25. Total RNA was extracted from 12 adenocarcinomas and mutations in TGF-beta RII were investigated by RT-PCR-restriction-SSCP analysis followed by sequencing analysis. Two out of 12 adenocarcinomas showed band shifts, indicative of mutations (16.7%). One was a CTG-to-TTG (Leu to Leu) transition at codon 308 without amino acid alteration and the other a frameshift deletion of one of two guanines at nucleotides 1434 to 1435 (codon 477 to 478). Semi-quantitative RT-PCR analysis demonstrated significantly reduced TGF-beta RII expression in adenocarcinomas, as compared with normal lung tissue. These results suggest that TGF-beta RII alterations may play a role in the acquisition of growth advantage by lung adenocarcinomas induced by BHP in rats.