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.     

Alkylation of rodent tissue DNA induced by N-nitrosobis(2-hydroxypropyl)amine.

Levels of methyl and hydroxypropyl adducts induced by single s.c. injections of various doses of tritium-labeled N-nitrosobis(2-hydroxypropyl)amine ([1-3H]BHP) were determined in the liver, pancreas, kidney and lung of hamsters and rats. At doses of BHP used in carcinogenesis studies (100-500 mg/kg), methylation of DNA was more extensive than its hydroxypropylation; however, it did not increase proportionally with the dose and gradually became secondary to hydroxypropylation at higher doses of the carcinogen. Ratios of hydroxypropyl versus methyl adducts also varied significantly depending on the tissue and species. In both species ratios of N7-hydroxypropylguanine (N7-HpG) versus N7-methylguanine (N7-MeG) were greater in kidney and pancreas than in liver or lung. Due to apparent differences in the repair of O6-methylguanine (O6-MeG) and O6-hydroxypropylguanine (O6-HpG), and the propensity of 2-hydroxypropylating as compared to methylating agents to yield a greater percentage of oxygen adducts, ratios of O6-HpG versus O6-MeG were markedly greater than those of N7-HpG versus N7-MeG. Levels of O6-HpG were greater than those of O6-MeG in rat liver, pancreas and kidney and also in hamster kidney, while such levels were similar in rat lung and also in hamster liver, pancreas and lung. Like N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP), BHP was activated primarily in the liver and induced substantially greater DNA damage in this than in any other tissue examined. However, unlike BOP and HPOP, which induced similar levels of hepatic DNA damage in the above two species, BHP methylated and hydroxypropylated hamster liver DNA more extensively than that of the rat. Differences between BOP and BHP were also observed regarding levels and distribution of DNA adducts in extrahepatic tissues. In rats, BHP induced greater levels of methylation and hydroxypropylation in lung than in kidney, while the reverse was observed with BOP. Apparently reduction of the beta-carbon of pancreas-specific nitrosamine carcinogens results in a shift of alkylation from kidney to the lung. Excretion of HPOP in the urine of BHP-treated animals and the observed saturation of DNA methylation at high doses of BHP, supported the hypothesis that the BHP-induced methylation of DNA proceeded via the intermediate formation of HPOP. This was further supported by the observation that both excretion of HPOP and levels of methyl adducts were greater in hamsters than in rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mutations of adenomatous polyposis coli and beta-catenin genes during progression of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine in rats

In the present study, we investigated mutations of the adenomatous polyposis coli (APC) and beta-catenin genes to clarify possible molecular mechanisms underlying development of lung tumors induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) in rats. Male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in drinking water for 12 weeks and then maintained without further treatment until sacrifice at week 25 DNA was extracted from paraffin-embedded tissues, and PCR-single-strand conformation polymorphism analysis, followed by nucleotide sequencing, was performed. No APC mutations were detected in 17 hyperplasias, but 2 of 15 adenomas (13.3%) and 8 of 20 adenocarcinomas (40.0%) showed changes within exon 1 to the mutation cluster region in exon 15. For beta-catenin, no mutations were detected in 17 hyperplasias, but 3 of 15 adenomas (20.0%) and 5 of 20 adenocarcinomas (25.0%) had alterations within or flanking codons corresponding to important phosphorylation sites. Immunohistochemical staining showed beta-catenin protein localized in the cell membranes in the surrounding normal-appearing lung and 216 hyperplasias and localized mainly in the cytoplasm and/or nucleus in 10 of 37 adenomas (27.0%) and 21 of 40 adenocarcinomas (52.5%). These results suggest that the APC-beta-catenin-T-cell factor signaling pathway is involved in the acquisition of growth advantage from adenomas to adenocarcinomas in BHP-induced rat lung carcinogenesis.     

Carcinogenicity of N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-oxopropyl)amine in MRC rats.

Weekly sc injections of equitoxic doses of N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitrosobis(2-oxopropyl)amine (BOP) to Wister-derived MRC rats induced tumors. The incidence, latency, multiplicity, morphologic type, and distribution of these tumors varied according to the compound given. The esophagus was the main target organ for BHP (100%), followed by the respiratory tract (87%), pharynx (80%), colon and liver (each 73%), kidneys (20%), thyroid gland (20%), and urinary bladder and urethra (each 7%). BOP was ineffective in the esophagus and pharynx but induced a higher incidence of tumors in the kidneys (27%), thyroid gland (60%), urinary bladder (33%), and urethra (73%) and fewer neoplasms in the respiratory tract (20%), colon (67%), and liver (53%). In addition, BOP caused a few, apparently primary, prostate squamous cell carcinomas. The results are compared with results of BHP treatment in Sprague-Dawley rats and with results of BHP and BOP treatment in Syrian golden hamsters.     

Increased expression of cyclooxygenase-2 protein in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine

The anti-inflammatory drugs, aspirin and piroxicam, are known to possess chemopreventive potential against rat superficial urinary bladder carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Recently, we found similar inhibitory effects with a selective cyclooxygenase (COX)-2 inhibitor, nimesulide. In order to clarify the inhibitory mechanisms, we have further studied the expression of COX-2 protein in urinary bladder tumors induced by BBN in Fischer 344 male rats. For comparison, papillomatosis caused by uracil-induced urolithiasis, and normal epithelial cells, were also investigated. Western blot analysis revealed COX-2 protein to be barely expressed in the normal epithelial cells, whereas it was increased 13-22-fold in varying sizes of urinary bladder tumors and 7-fold in papillomatosis. Immunohistochemically, COX-2 protein was diffusely expressed in transitional cell carcinomas and nodulo-papillary hyperplasia but weakly expressed only in basal cells in simple hyperplasia and normal-looking surrounding epithelia. In papillomatosis, it was moderately expressed only in endothelial cells in stroma. These results indicate that COX-2 plays important roles in the development of preneoplastic and neoplastic lesions in the rat urinary bladder, and therefore could be a good target for chemoprevention of superficial lesions.     

Determination of N-nitrosobis(2-hydroxypropyl)amine in environmental samples

N-Nitrosobis(2-hydroxypropyl)amine (ND2HPA) is a potent pancreatic carcinogen in hamsters and induces gastrointestinal and respiratory tract cancer in rats. The precursor amines, diisopropanolamine (Di-PA) and triisopropanolamine (Ti-PA), are used in some manufacturing processes and in cosmetic preparations. We have found low levels of ND2HPA in commercial Ti-PA (21-270 ng/g) and in Di-PA (20-1 300 ng/g) and have demonstrated that ND2HPA is formed from Ti-PA and nitrite in a yield comparable to that observed for formation of N-nitrosodiethanolamine (NDELA) from triethanolamine under relatively mild conditions. After reaction for 4 h at 37 degrees C (10 mmol/L amine, 40 mmol/L nitrite, pH 3.0), the ND2HPA yield was 0.51%. The NDELA yield under the same conditions was 0.96%. ND2HPA was determined by gas chromatography-thermal energy analysis (GC-TEA) and GC-high-resolution mass spectrometry (GC-MS) selected ion monitoring of the tert-butyldimethylsilyl (t-BDMS) ether after extraction on a Celite 560 column. The t-BDMS ethers of ND2HPA and NDELA yielded intense, structurally significant peaks at m/z 333.2030 and 305.1716, respectively. The GC-MS procedure provides sensitivity and selectivity comparable to that of GC-TEA.     

Ki-ras activation in pancreatic carcinomas of Syrian hamsters induced by N-nitrosobis(2-hydroxypropyl)amine.

Five pancreatic carcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in Syrian golden hamsters were analyzed for activation of Ki-ras at codons 12 and 13, using the polymerase chain reaction and direct sequencing. The Ki-ras gene was shown to be activated in four of the five carcinomas, and the results were further confirmed by subcloning and sequencing. All the mutations involved a G-to-A transition at the second position of codon 12, which resulted in a change at the amino acid level from glycine to aspartic acid. This mutation is identical with that reported for pancreatic tumors of Syrian hamsters induced by N-nitrosobis(2-oxopropyl)amine.     

Initiation activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine in the rat liver

The initiation potential of N-nitrosobis(2-hydroxypropyl)amine (NDHPA) endogenously synthesized from bis(2-hydroxypropyl)amine (DHPA) or tris(2-hydroxypropyl)amine (THPA) in the presence of sodium nitrite (NaNO2) was investigated in the rat liver by quantitation of hepatocellular foci showing phenotypic expression of glutathione S-transferase placental form (GST-P). The investigation consisted of two experiments. In the first, male Wistar rats were divided into six groups as follows: group 1 was non-treated; groups 2 and 3 received 0.15% and 0.3% NaNO2, respectively; group 4 received 1% DHPA; groups 5 and 6 received 1% DHPA together with 0.15% and 0.3% NaNO2, respectively. In experiment 2, the same protocol was used except that 2% THPA was substituted for 1% DHPA. The treatments were continuous until sacrifice at week 94 in experiment 1 and week 104 in experiment 2. As a result putative preneoplastic GST-P-positive foci observed in the liver and increased dose-dependently in rats from groups 5 and 6 which had received DHPA and NaNO2, but not in rats administered THPA and NaNO2. The results indicate that endogenously synthesized NDHPA from DHPA and NaNO2 is capable of initiating neoplastic development in the rat liver.     

Distribution and metabolism of N-nitrosobis(2-hydroxypropyl)amine in rats

The metabolic fate of the lung carcinogen N-nitrosobis(2-hydroxypropyl)amine (ND2HPA) in male Wistar rats was studied. The blood level after a single intraperitoneal (i.p.) injection of [1-14C]-ND2HPA at a dose of 3 g/kg body weight reached a maximum within 1 h. Most of the administered 14C was eliminated via the urine; 90.8% of the 14C was excreted in urine within 24 h, 5.5% in faeces, and 3.2% in expired air. About 11% of the 14C was detected in bile collected over 24 h. A relatively high concentration of 14C was found in the blood and target organs, such as the lung, liver, thyroid gland and kidney 1 h after treatment. Analysis by high-pressure liquid chromatography showed that the 14C in the blood and urine was mostly accounted for by unchanged ND2HPA, together with smaller amounts of N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (N2HP2OPA). ND2HPA and N2HP2OPA were also detected in the lung and liver of rats 30 min to 12 h after the administration and were present in higher concentrations in the blood and lung than in the liver and pancreas. Besides ND2HPA and N2HP2OPA. N-nitrosomethyl(2-hydroxypropyl)amine (NM2HPA) was also found in urine collected over 6 h. ND2HPA, N2HP2OPA and NM2HPA showed mutagenicity in the Salmonella assay system with metabolic activation by a 9000 X g supernatant of rat liver, and N2HP2OPA was also mutagenic in the presence of a rat lung preparation. These data suggest that N2HP2OPA and NM2HPA might be important intermediates in the metabolic activation of ND2HPA to its ultimate carcinogenic form 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.