The anomalous biological activity of nitroso-2-oxopropyl compounds

The carcinogenic action of a set of N-nitroso compounds containing the 2-oxopropyl group was considered in relation to their metabolism and their activity as alkylating agents for DNA. In contrast with the great carcinogenic potency of methylnitrosourea and ethylnitrosourea, comparable with the corresponding dialkylnitrosamines, 2-oxopropylnitrosourea is a weak carcinogen with a limited range of target organs in rats and hamsters. 2-Oxopropylnitrosochloroethylurea was somewhat weaker than 2-oxopropylnitrosourea and similarly induced spleen hemangiosarcomas in hamsters, but few tumors of any kind in rats. The relatively much more potent carcinogenicity of nitrosobis-(2-oxopropyl)amine, nitroso-(2-hydroxypropyl) (2-oxopropyl) amine and methylnitroso-2-oxopropylamine suggests that the activity of an oxopropylating agent is not involved in carcinogenesis by nitroso-2-oxopropylamines. The nitrosamines are likely to undergo extensive metabolism to form proximate carcinogenic moieties, probably including the methyldiazonium ion, which are responsible for the induction of a broad range of tumors in rats and hamsters. These include tumors of the liver, pancreas ducts, lung and nasal mucosa in hamsters, and esophagus, liver, lung, thyroid, kidney, trachea, bladder and nasal mucosa in rats.     

Effect of beta-oxidized nitrosamines on syrian hamsters. III. 2,2'-Dihydroxydi-n-propylnitrosamine.

2, 2-Dihydroxy-di-n-propylnitrosamine (DHPN), an assumed metabolite of di-n-propylnitrosamine (DPN), injected subcutaneously once weekly for life, was carcinogenic in Syrian hamsters. The main target organs were the respiratory tract, pancreas, liver, and kidneys. In the respiratory system the most affected segments were the nasal cavities and the lungs. Adenomas and adenocarcinomas, mostly of ductal origin, were induced in the pancreas. Liver neoplasms were hemangloendotheliomas, angiosarcomas, hepatocellular adenomas, cholangiomas, and cholangiocarcinomas. Kidney neoplasms were adenomas and adenocarcinomas. The morphology of the induced neoplasms was described, as well as the effects of DHPN, compared to those another possible metabolite of DPN, 2-hydroxypropyl-n-propylnitrosamine (2-HPPN), which is formed in vivo with only 1 aliphatic chain degraded via theta-oxidation.     

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is an active transplacental carcinogen in Syrian golden hamsters

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in adult laboratory rodents. Our previous studies have shown that NNK crosses the placenta in pregnant hamsters and is metabolized into DNA-methylating and clastogenic intermediates by fetal respiratory tract tissues. Based on these findings, we have tested the transplacental carcinogenicity of NNK in this species. Groups of pregnant hamsters were given s.c. injections of single or multiple doses of NNK (cumulative dose range, 50-300 mg/kg) on day 15 (last day of gestation) or on days 13, 14, and 15 of gestation. Within 1 year after treatment, up to 70% of the offspring developed tumors in various organs, including respiratory tract, nasal cavity, adrenal glands, pancreas, and liver. No tumors were found in the control hamsters treated with the vehicle (trioctanoin) alone. The overall tumor incidence was proportional to the cumulative dose. Females had a generally higher tumor incidence than males.     

The role of N-(nitrosomethylamino)propionitrile in betel-quid carcinogenesis

N-(Nitrosomethylamino)propionitrile (NMAP) was isolated and identified in the saliva of betel-quid chewers in amounts ranging from 0.5 to 11.4 micrograms/l. Groups of 21 male and 21 female rats were given 60 subcutaneous injections of NMAP over a 20-week period (total doses, 0.055 and 0.23 mmol/rat). After 106 weeks, the higher dose had induced 18 (86%) malignant tumours of the nasal cavity in male and 15 (71%) in female rats. Nine (43%) liver tumours were observed among animals treated with the lower dose. Fischer 344 rats were treated with a single dose of NMAP (intravenously or subcutaneously, 0.4 mmol/kg; or by swabbing the oral cavity, 2.21 mmol/kg), and the levels of N7-methylguanine (7-meG) and O6-methylguanine (O6-meG) were measured in DNA isolated from oesophagus and nasal mucosa, which are target organs, and from liver which is not. Higher levels of O6-meG and 7-meG were detected in the nasal mucosa and lesser DNA methylation in the liver and oesophagus, independent of the mode of administration. This correlates with the results of the study of the tumorigenic properties of NMAP in rats.     

Contrasting carcinogenic effects of nitroso-2,6-dimethylmorpholine given by gavage to F344 rats and Syrian golden hamsters

Nitroso-2,6-dimethylmorpholine (Me₂NM) has been given by gavage to F344 rats of both sexes and to male Syrian hamsters at similar doses. The rats died more rapidly from the tumors induced than did the hamsters, the males being more susceptible than the females. The rats developed mainly carcinomas and papillomas of the esophagus, whereas the hamsters died with angiosarcomas of the liver, ductal adenocarcinomas of the pancreas, adenocarcinomas of the lung and olfactory adenocarcinomas of the nasal cavity. It was notable that, with the exception of a few lung tumors in the rat, none of the tumors found in the hamster were induced by Me₂NM in the rat, and no esophageal tumors were seen in the hamsters.     

Alkylation of DNA related to organ-specific carcinogenesis by N-nitroso compounds.

Alkylation of DNA by a number of methylating and ethylating carcinogens, mainly N-nitroso compounds, has been examined in target and non-target organs of rats and Syrian hamsters. Six hours after administration by gavage of small doses identical to those given twice weekly for several months to elicit tumours, animals were killed and dissected. DNA was isolated from several organs and hydrolysed, and the content of methyl- and ethylguanines was measured using high-performance liquid chromatography for separation. In most experiments, radiolabelled carcinogen was used, but in some cases measurement of alkylguanines was by fluorescence. Methylation, O6- and N7-, by methylating compounds was much more extensive than ethylation by the corresponding ethyl compounds, irrespective of their relative potencies in inducing tumours. Similar patterns of alkylation were found in target organs and in non-target organs of the carcinogens. Only marginal differences in methylation were seen with N-nitro-sobis(2-oxopropyl)amine between male and female rat livers, although liver tumours are induced only in females, in feminized males and in old males. Deuterium labelling of the methylene of N-nitrosoethylmethylamine had little effect on methylation or ethylation of DNA in rat liver, although the deuterated compound was a much more potent liver carcinogen. The conclusion is that reactions of the carcinogen other than alkylation of DNA are important in giving rise to tumours.     

Species and organ differences in DNA adduct formation and repair after treatment with 4-hydroxyaminoquinoline 1-oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

Species and Organ Differences in DNA Adduct Formation and Repair after Treatment with 4-Hydroxyaminoquinoline 1-Oxide

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.     

Lysosomal changes and enhanced metastatic growth: an experimental study of the effects of some non-ionic surfactants

The effects on metastatic growth of Triton WR-1339 and three chemically related non-ionic detergents have been investigated in 40 hamsters bearing subcutaneous transplants of a metastasizing lymphoma (ML). Ten further untreated animals with tumour grafts and 20 normal animals without tumour grafts acted as controls. In hamsters without tumours, the four detergents commonly produced three hepatic changes: an increase in the liver/body weight ratio, an increase in stainable acid phosphatase, and an increase in the number and size of the lysosomes, together with ultrastructural changes. In tumour-bearing animals, the detergents all produced a paradoxical effect, partially inhibiting growth of the tumour graft but markedly facilitating metastatic spread. More organs contained secondary tumours in the test groups, and the pattern of involvement in certain organs (liver, kidneys) was strikingly altered, with diffuse rather than focal metastatic growth. Detailed investigations of the liver established that treated animals all showed the same (but more profound) changes as those seen in detergent-treated hamsters without tumours-i.e., high liver/body weight ratios, high acid phosphatase scores, and lysosomal damage. No lysosomal lesions were found in untreated tumour-bearing hamsters. In the discussion, it is stressed that the detergents tested acted principally on host (cf. tumour) tissues, and it is suggested that local lysosomal damage in lysosome-rich organs such as the liver may be one factor whereby these detergents facilitate local tumour spread. Ways in which lysosomal lesions may affect local tumour growth are discussed, and analogies are drawn from previous work relating to lysosomal changes and the (associated) enhanced growth of mycobacteria. The implication of the present findings in relation to the old “soil” hypothesis of metastatic growth are indicated but the fallacies of premature generalizations about lysosomal changes and metastasis are stressed.     

Cyanoethylation of DNA in vivo by 3-(methylnitrosamino)propionitrile, an Areca-derived carcinogen

2-Cyanoethyldiazohydroxide is a likely product of metabolic alpha-hydroxylation of 3-(methylnitrosamino)propionitrile (MNPN). The reaction of 2-(N-carbethoxy-N-nitrosamino)propionitrile, a stable precursor of 2-cyanoethyldiazohydroxide, with deoxyguanosine, catalyzed by porcine liver esterase, was investigated. Two major deoxyguanosine adducts were produced. They were isolated by high-performance liquid chromatography and characterized by their UV spectra, mass spectra, and proton magnetic resonance spectra. On the basis of these spectral data, the structures of the two adducts were assigned as 7-(2-cyanoethyl)guanine and O6-(2-cyanoethyl)deoxyguanosine. The potential of MNPN to cyanoethylate DNA in F344 rats was evaluated by measuring 7-(2-cyanoethyl)guanine and O6-(2-cyanoethyl)guanine in the liver, nasal mucosa, and esophagus. The highest levels were detected in the nasal cavity, which is one of the major target organs for the carcinogenic effects of MNPN.     

Spontaneous tumors and common diseases in two colonies of Syrian hamsters. II. Respiratory tract and digestive system.

Spontaneous respiratory tract neoplasms in Syrian hamsters occurred in almost equal frequencies in two colonies: 3% in the Eppley Colony (EC) and 3.6% in the Hannover Colony (HC). Neoplasms were in the nasal cavity, trachea, and lungs, and most were benign; however, 2 adenocarcinomas of the nasal cavity (EC) and 1 adenocarcinoma of the larynx (HC) were found. The incidence of digestive tract tumors showed a more marked difference than that of the respiratory tract: 7% in the EC and 23% in the HC. Digestive tract tumors accounted for 15 and 41% of all tumors in the EC and HC, respectively. All EC digestive tract neoplasms were benign and occurred mostly in males; 19 (83%) were forestomach papillomas and the remaining 4 )17%) were liver hemangioendotheliomas (2) and pancreatic duct adenomas (2). In the HC, almost 50% of the digestive tract neoplasms were malignant and most frequent in females. These tumours include 19(42%) intestinal adenocarcinomas, 18(40%) liver neoplasms (hemangioendotheliomas, cholangiomas, cholangiocarcinomas), 5 (13%) forestomach papillomas, and 2 (4%) gallbladder polyps. The morphology of these neoplasms was reported.     

Carcinogenicity of N-nitrosomethyl(2-oxobutyl)amine and N-nitrosomethyl-(3-oxobutyl)amine in Syrian hamsters with special reference to the pancreas

Studies with oxidized derivatives of N-nitrosodi-n-propylamine suggested a structure-activity relationship between pancreatic cancer induction in Syrian hamsters and position and degree of nitrosamine oxidation. To elucidate the importance of the position of the oxidized substituent relative to the N-nitroso group in pancreatic carcinogenesis, we compared the toxicity and carcinogenicity of two substituted methylbutylnitrosamines. N-Nitrosomethyl(2-oxobutyl)amine (M-2-OB) and N-nitrosomethyl(3-oxobutyl)amine (M-3-OB) were given in equitoxic doses to male and female Syrian hamsters. The 50% lethal doses for M-2-OB and M-3-OB in males and females, respectively, were 92 and 160 and 705 and 810 mg/kg body weight. M-2-OB, although given in significantly smaller doses (minimum dose, 2.3 mg/kg body weight) than was M-3-OB (minimum dose, 17.6 mg/kg body weight), induced a much broader spectrum of neoplasms (in 17 tissues), whereas M-3-OB induced tumors in only 5 tissues and had no carcinogenic effect in the pancreas. M-2-OB, however, produced pancreatic ductular-ductal adenocarcinomas in over 90% of the males and 67% of the females, even at the lowest doses (2.3 and 4.0 mg/kg, respectively). Although both compounds caused a similar incidence of morphologically equivalent neoplasms (mostly adenocarcinomas) in the nasal and paranasal cavities, the remaining distribution of affected tissues differed significantly. M-2-OB predominantly affected the lip (epitheliomas, squamous cell carcinomas), liver (cholangiomas and cholangiocarcinomas), and flank organ (epitheliomas, squamous cell carcinomas). The principal target organs for M-3-OB were the cheek pouch (papillomas, squamous cell carcinomas) and trachea (polyps). In contrast to M-2-OB, M-3-OB did not induce renal and urethral tumors. These findings indicate the importance of the 2-oxo group as a prerequisite for the carcinogenicity of methylalkylnitrosamines in the hamster pancreas; however, a methyl group in one aliphatic chain, alpha to the N-nitroso function, appears to cause the molecule to lose its selectivity for the pancreas.     

Comparative study of the carcinogenic effect of BHP and BAP on NMRI mice

The carcinogenic effects of N-bis(2-hydroxypropyl)nitrosamine (BHP) and N-bis(2-acetoxypropyl)nitrosamine (BAP) were studied in NMRI mice treated subcutaneously once weekly for life. The highest incidences of tumours were found in the lung (73–100%), in the liver (67–100%) and nasal cavity (0–33%) in both sexes of mice. Data were similar in animals treated with BHP or BAP. These tumours were adenomas, adenocarcinomas and squamous cell carcinomas (nasal cavity and lung) as well as hemangioendotheliomas and hemangioendotheliosarcomas (liver). The lung tumours were found in the control mice (27–50%) 20 weeks later than in the experimental animals. The rate of malignant neoplasms rose with increasing survival and decreasing dose levels.     

Carcinogenesis by nitrosomorpholines, nitrosooxazolidines and nitrosoazetidine given by gavage to Syrian golden hamsters

Five cyclic nitrosamines, four containing oxygen in the ring,were administered by gavage to groups of 20 male Syrian goldenhamsters. After administration of very similar doses, nitrosomorpholine,nitroso-2-methylmorpholine and nitroso-5-methyl-1,3-oxazolidinecaused the animals to die with tumors after similar times, butnitrosomorpholine induced mainly tumors of the nasal cavity(and a few of the trachea), whereas the 2-methyl derivativeinduced tumors of the nasal cavity and liver. While nitroso-1,3-oxazolidineand its 5-methyl derivative both induced liver tumors (but notumors in the nasal cavity) those induced by the former compoundtook much longer to kill the animals. Nitrosoazetidine, a livercarcinogen in rats, but which had been reported to be inactivein hamsters, did induce tumors of the liver in 30% of hamstersafter a much larger dose than the other cyclic nitrosamines.     

Carcinogenic effects of different nitroso-compounds in chinese hamsters: N-dibutylnitrosamine and N-nitrosomethylurea.

Three hundred and twenty Chinese hamsters (Cricetulus griseus) (CH) were subcutaneously (s.c.) treated with 1/5, 1/10 or 1/20 the mean lethal dose (LD50) of N-dibutylnitrosamine (DBN) or N-nitrosomethylurea (NMU). In several respects these two substances produced the same organotropy in the CH as in the Syrian golden (SGH) and European hamsters (EH). DBN prolonged lung tumours and neoplasms of the injection site. NMU induced tumours of the injection site only.     

Carcinogenic effect of 2,2'-dimethyldipropylnitrosamine in Syrian hamsters.

Oxidation at the beta carbon occurred in metabolism of di-n-propylnitrosamine (DPN), previously shown to be carcinogenic for animals. When 2,2'-dimethyldipropylnitrosamine (DMDPN) was injected sc once a week for life into male and female Syrian hamsters at levels of 500, 250, 125, and 62.5 mg/kg body weight, it induced neoplasms in the nasal cavities, larynx, trachea, and stem bronchi. Since the presence of a methyl group on the beta carbon suggested that DMDPN could not undergo beta oxidation, the carcinogenicity of DPN for these portions of the respiratory tract was probably unrelated to beta oxidation, though earlier experiments had indicated the possibility of this mechanism. Because DMDPN failed to induce neoplasms in other organs, the carcinogenicity of DPN or its beta metabolites for the lungs, liver, pancreas, and kidneys was not explained by this experiment.     

Effects of vitamin C on tumor induction by diethylnitrosamine in the respiratory tract of hamsters exposed to cigarette smoke

A large dietary supplement (1%) of vitamin C was given to male Syrian hamsters exposed to cigarette smoke and receiving 12 weekly subcutaneous injections of diethylnitrosamine (DEN) to determine whether or not high doses of vitamin C can prevent the development of tumors by DEN in the respiratory tract. Treatment with DEN developed various types of tumors in the respiratory tract of hamsters and cigarette smoke exposure potentiated the tumor development in the nasal cavity, larynx and trachea. In comparison with the smoke-exposed hamsters treated with DEN, the vitamin C-supplemented hamsters showed significantly lower incidence of nasal cavity tumors and exhibited significantly earlier appearance of tracheal tumors. In addition, the laryngeal tumors also tended to develop earlier in the vitamin C-supplemented hamsters. The results indicate that high doses of vitamin C may inhibit tumor induction by DEN and exposure to cigarette smoke in the nasal cavity, but the development of laryngeal and tracheal tumors appeared to be accelerated by vitamin C supplement.     

A study of tobacco carcinogenesis XLVIII. Carcinogenicity of N'-nitrosonornicotine in mink (Mustela vison).

During tobacco processing and smoking, nicotine and nornicotine give rise to N'-nitrosonornicotine (NNN), a highly abundant, strong carcinogen. NNN is known to exert carcinogenic activity in mice, rats and hamsters. Major target organs for NNN carcinogenicity in the rat are the esophagus and the nasal mucosa, and in the Syrian golden hamster trachea and nasal mucosa. In comparison with the rat, the mink (Mustela vison) has a markedly expanded nasal mucosa. Therefore, we explored in this study whether the mink could serve as a non-rodent model for nasal carcinogenesis using NNN as the carcinogen. Twenty random-bred mink, beginning at the age of 3 weeks, received twice weekly s.c. injections of NNN, a total dose of 11.9 mM per animal over a 38 week period. All of the 19 mink at risk developed malignant tumors of both the respiratory and the olfactory region of the nose within 3.5 years. In most animals the malignant tumors, primarily esthesioneuroepithelioma, invaded the brain. Remarkably, NNN induced no other tumors in the mink. None of the control animals developed nasal tumors nor tumors at other sites during the 3.5 years of the assay. The historical data from the farm did not reveal any spontaneous occurrence of nasal tumors in mink at any age. This study supports the concept that NNN is a proven carcinogen for multiple species of mammals and that the mink can serve as a non-rodent, non-inbred animal model for nasal carcinogenesis, especially since NNN induces only tumors in the nasal cavity in this species and not at other sites, as it does in mice, rats and hamsters.     

Cell specific DNA alkylation in target and non-target organs of N-nitrosobis(2-oxopropyl)amine-induced carcinogenesis in hamster and rat

Tissue-specific formation and short-term persistence of alkylated DNA bases have been studied immunocytochemically in Syrian hamsters and rats killed 3-48 h after a single s.c. or oral dose of N-nitrosobis(2-oxopropyl)amine (BOP). Antisera specific for O6-(m)ethylguanine and for 7-(m)ethylguanine were used. Strong nuclear staining, indicative of a high level of DNA alkylation, was observed at all time points in the intra- and interlobular duct cells and in the centroacinar cells of the hamster pancreas, the main target organ of BOP-induced carcinogenesis. Acinar cells were weakly stained for up to 24 h. In the liver, nuclear staining was strong in all cell types, and more pronounced in the periportal than in the central venous area. Both O6-alkylguanine and 7-alkylguanine preferentially disappeared from the centrilobular area of the liver which is in agreement with the high O6-methyltransferase activity of liver and the unusually high levels of 7-methylguanine DNA glycosylase activity in hamster tissues. Strong staining was observed throughout the experiment in the tubular cells of the renal cortex and in bronchiolar Clara and alveolar type II cells of the lung. The staining intensity of the cells of the thyroid follicles and of the columnar epithelial cells of the colon was moderate. In the rat, nuclear staining was strong in the nasal cavity (Bowman glands), the epithelium lining the thyroid follicles, the lung, liver and in the fibroblasts of the ureter intima and adventitia. The epithelial cell nuclei of the colon and ureter were moderately stained. In the pancreas, staining was weak in acinar, duct and islet cells; no acinar staining remained at 48 h. In the liver, nuclear staining was strong all over the lobule. O6-Alkylguanine was preferentially removed from the centrilobular area. The renal tubular cells were only weakly stained. From the present study we can conclude that--with the exception of hamster kidney and rat liver--high levels of DNA alkylation and stability of the alkylated products were related to a high tumor incidence.     

Comparative carcinogenicity in F344 rats and Syrian golden hamsters of N′-nitrosonornicotine and N′-nitrosonornicotine-1-N-oxide

N′-Nitrosonornicotine (NNN) or N′-nitrosonornicotine-1-N-oxide (NNN-1-N-oxide), one of its metabolites, was added to the drinking water (0.012% for 36 weeks) of groups of male and female F344 rats or to the drinking water (0.016% for 31 weeks) of groups of male and female Syrian golden hamsters. All rats treated with NNN had died after 12 months but 50% of those treated with NNN-1-N-oxide survived for 22 months. NNN induced esophageal tumors in 23/24 rats and nasal cavity tumors in 21/24 rats. NNN-1-N-oxide induced esophageal tumors in 10/24 rats and nasal cavity tumors in 18/24 rats. There was no difference in survival rates among hamsters treated with either NNN or NNN-1-N-oxide. NNN induced tracheal tumors in 2/20 hamsters and nasal cavity tumors in 4/20 hamsters. NNN-1-N-oxide did not induce respiratory tract tumors in hamsters. These results demonstrate that NNN-1-N-oxide is less carcinogenic than NNN in F344 rats and Syrian golden hamsters.