A dose-response study in carcinogenesis was carried out with .Vnitrosomorpholine in female F344 rats. The compound was administered in drinking water, which was supplied in controlled amounts of 20 ml per day per rat, 5 days a wk. At the two highest dose rates, 100 mg/liter and 40 mg/liter, treatment lasted 25 and 40 wk, respectively. At the other dose rates, which differed by a factor of 2.5, treatment lasted 50 or 100 wk. The average total dose received by each rat ranged from 250 mg to 0.7 mg. There were 100 animals per group at the lowest dose rates and 24 animals per group at the highest dose rates. Total doses of nitrosomorpholine above approximately 30 mg per rat caused a statistically significant decrease in survival, but at lower doses survival was similar to that of untreated controls. In nearly all of the treated groups there was a statistically significant increase in the incidence of benign or malignant hepatocellular neoplasms, with a highly significant dose-related trend. At the higher doses there was a significant incidence of hemangiosarconias of the liver. Both hepatocellular carcinomas and hemangiosarcomas metastasized to the lungs and other organs. At the highest doses there was a significant incidence of neoplasms of the tongue and esophagus, which were rarely seen at the lower doses. The results suggest that even the lowest dose of nitrosomorpholine received by the rats, 0.7 mg or approximately 3 mg/kg body weight, was not a no-effect dose during the 2-yr lifetime of a rat. Probit analysis of the results indicate a dose estimated to cause tumors in 50% of the population of 25 mg nitrosomorpholine for liver neoplasms.

Morphology and development of experimental bronchiolar lung tumors were studied in Syrian hamsters, using light and electron microscopic techniques. At the age of 9 weeks, 46 hamsters were each given one weekly gavage of 6.8 mg N-nitrosomethyl-n-heptylamine for 35 weeks, and hamsters were examined at intervals from 2 to 46 weeks. The present report describes the progression of adenocarcinomas of bronchiolar cell origin to adenosquamous and squamous cell carcinomas. Squamous metaplasia was commonly noted at the tumor periphery, i.e., zone of growth. In 20 hamsters, 22 adenosquamous and two squamous cell carcinomas (one a large cell carcinoma) were diagnosed by light microscopy. Overt keratinization was infrequent. Squamous cell metaplasia was not a feature of papillary neoplasms but was seen mainly with acinar structures. Ultrastructurally, squamous differentiation (metaplasia) appeared to develop along two different pathways. First, secretory cells were observed with large numbers of intermediate filaments and tonofilaments, with concurrent loss of organelles such as secretory granules and microvilli. Second, squamous metaplasia also appeared to develop from a progeny of tumor cells that failed to mature into secretory cells. Such cells were often present within the basal layer of secretory acini and resembled basal cells of the tracheobronchial tree. These observations were supported by increased expression of cytokeratins, as revealed by immunohistochemical procedures. Immunoelectron microscopic examination localized hamster Clara cell antigen in secretory granules of neoplastic Clara cells, in the cytoplasm between granules, and at the microvillous border. With the onset of squamous differentiation, Clara cell antigen was progressively lost from secretory cells and was only rarely seen in cells with tonofilaments. No labeling was present in squamous cells arising at the base of tumor acini. These results suggest that pulmonary squamous cell carcinomas may develop by direct squamous differentiation of secretory cells or may proceed from undifferentiated tumor cells.

The incidence of a set of neoplasms arising "spontaneously" in Fischer 344 (F344) rats was determined in control and carcinogen-treated animals. Data were obtained from approximately 9000 rats (4000 males and 5000 females) used to study the carcinogenicity of a variety of alkylating compounds, including N-nitroso compounds, azoxyalkanes, and triazenes. In these experiments treated rats and controls were allowed to die naturally and were necropsied, and the tissues were examined histopathologically. The spontaneous neoplasms of interest were mononuclear cell leukemia and neoplasms of the anterior pituitary, adrenal medulla, pancreas, thyroid gland, mammary gland, and testis. These tumors were generally absent from control animals that (rarely) died before 70 wk of age. Although many carcinogen-treated rats died early with treatment-related tumors, a substantial number (1700 males and 2300 females) survived as long as controls. The incidence of spontaneous neoplasms was determined among controls and chemically treated rats at 10-wk intervals from 0 to 140 wk. The incidence of spontaneous tumors was not higher and was frequently statistically lower among treated rats than the corresponding incidence in controls, with the exception of leukemia in female rats. The same result was obtained with the subset of carcinogens not requiring metabolic activation (mostly alkylnitrosoureas). These data indicate that in this rat tumor model system, the alkylating carcinogens, while capable collectively of tumor induction at more than 20 sites, did not accelerate the development of any of the six spontaneously arising solid tumors. This suggests that these spontaneous tumors might arise by a mechanism that is unresponsive to the actions of the alkylating carcinogens.

Two nitrosamines derived from nitrosation of piperazine, 1-nitrosopiperazine (NO-PIP) and 1,4-dinitrosopiperazine (DNP), were administered to groups of twelve female F344 rats intravesically. The doses were, respectively, 40 mg and 5.2 mg twice a week for 48 and 36 weeks in aqueous solution. Ten DNP-reated animals survived the treatment; six had tumors related to the treatment, nasal mucosa adenocarcinomas or neuroblastomas in five and a transitional cell carcinoma of the bladder in one. Rats treated with NO-PIP received a ten times greater dose, and all died by week 59, two with transitional cell neoplasms of the bladder and four with carcinomas of the nasal mucosa. NO-PIP was probably in part converted by transnitrosation to DNP. Piperazine, widely used as an oral anti-helminthic, could interact with nitrosating agents in vivo to form the two nitrosamines here shown to pose a possible carcinogenic risk if present in the bladder, by absorption through the bladder wall.

Metabolites produced by enzymic oxidation are believed to be responsible for the mutagenicity and carcinogenicity of N-nitrosamines. Although alpha-hydroxy compounds are often considered, a related and more stable oxidation product, the alpha-carbonyl compound, was studied here. The alpha-carbonyl derivatives of nitrosodimethylamine (NDMA) and ethylnitrosomethylamine (oxidized at either the methyl or the ethyl group) were synthesized. The derivatives were methylnitrosoformamide (MNFA), ethylnitrosoformamide (ENFA) and methylnitrosoacetamide (MNAA). These compounds were then studied as potential toxic, mutagenic and carcinogenic intermediates. All three compounds were very potent directly acting mutagens to Salmonella typhimurium TA1535. Mutational Fingerprints in Escherichia coli of MNFA and ENFA (but not MNAA) matched those produced by SN1-type methylating and ethylating compounds respectively. The latter results indicate that the two alkylnitrosoformamides could be intermediates in the mutagenicity of the parent nitrosamines. In animal studies the putative metabolite MNFA was more acutely toxic than NDMA in F344 rats. In chronic experiments with MNFA in F344 rats and Syrian golden hamsters, tumors of the forestomach were induced by oral administration in most animals (except female hamsters) within 8 months. The properties of these oxidized derivatives of N-nitrosamines are consistent with expectations for proximate carcinogenic intermediates.

Histogenetic features of lung tumours were studied in Syrian hamsters that had been induced with 6.8 mg N-nitrosomethyl-n-heptylamine/animal by gavage once a week for 35 weeks. At intervals from experimental week 2 until week 46, pulmonary tissues from hamsters were examined by light and electron microscopy. This report describes early hyperplastic lesions associated with terminal bronchioles and the progression of these lesions to bronchioloalveolar tumours. Using immunohistochemical and ultrastructural colloidal gold labelling techniques, hamster Clara cell antigen was found to be localized in Clara cell granules and smooth endoplasmic reticulum of normal cells, in dysplastic Clara cells migrating through basement membrane defects or from the open end of terminal bronchioles, and in hyperplastic peribronchiolar cell foci. The latter progressed to bronchioloalveolar tumours growing out along alveolar basement membranes in a characteristic lace-like, lepidic pattern. Tumours were composed of secretory (Clara), ciliated, mucous, and undifferentiated cells, as well as trapped, non-neoplastic alveolar type II cells. Hyperplastic neuroendocrine cell foci lining airways were immunoreactive for chromogranin, but these cells did not participate in the pre-neoplastic or neoplastic process. It is suggested that bronchioloalveolar carcinomas in hamsters are derived from bronchiolar secretory (Clara) cells growing along alveolar walls, differentiating into other bronchiolar cell types and entrapping resident alveolar type II cells. Due to the migratory capacity of Clara cells, it is also possible for tumours composed of bronchiolar cells to develop at the lung periphery.

Carcinogenic alkylating agents administered orally are metabolized in the liver and alkylate DNA in liver cells of rats and hamsters. They frequently, but not always, induce liver tumors, in addition to tumors of other organs. Directly acting alkylating agents, such as alkylnitrosoureas and alkylnitrosocarbamates, rarely induce liver tumors, although they alkylate DNA in liver cells. The methylating agents nitrosodimethylamine and azoxymethane induce high incidences of liver tumors in rats when given in drinking water, but few or no liver tumors when given by gavage, although the total dose and the weekly dose were the same in either regimen. In contrast, methylnitrosoethylamine and nitrosodiethylamine give rise to liver tumors in rats in high incidences whether given by gavage or in drinking water. Sharp differences are also observed with other nitrosamines, such as those containing a propyl group with an oxygen substituent in the 2-position. These discrepancies indicate that, in addition to alkylation of DNA, pharmacokinetics of dosing and distribution and other reactions of the carcinogen are the dominant factors in determining the development of tumors in the liver.

Two N-nitroso compounds that are derivatives of N,N-dimethylethylenediamine and are therefore strongly basic, were tested for carcinogenic activity. They were methylnitrosamino-N,N-dimethylethylamine (MNDMEA) and N,N-dimethylaminoethylnitrosoethylurea (DMENEU). Each was administered orally to male and female F344 rats by gavage. MNDMEA was also given by gavage to Syrian hamsters and to rats as a solution in drinking water. The response of rats treated with MNDMEA was almost the same by the two modes of treatment and all developed tumors of the esophagus and died in less than 40 weeks; many also had tumors of the nasal mucosa. Hamsters were less susceptible to the nitrosamine than rats, since they survived longer following a larger dose and the tumor incidence was small; several hamsters had tumors of the nasal mucosa, some males also had tumors of the liver and lung and one male and two females had a tumor of the colon. Although it is a strong directly acting mutagen, dimethylaminoethylnitrosoethylurea was weakly carcinogenic in rats, giving rise to tumors of the uterus and mammary gland in females, but having no particular target organ in male rats. The presence of a basic center in these N-nitroso compounds does not prevent their absorption nor their entry into cells, which they can transform to tumors.

Glossary Foreword Acknowledgements 1. Introduction 2. Occurence, formation and detection of N-nitroso compounds 3. Chemical properties of N-nitroso compunds 4. Metabolism and cellular interactions of N-nitroso compunds 5. Toxicity of N-nitroso compunds 6. Mutagenesis and cell transformation by N-nitroso compunds 7. Structure-activity relations in carcinogenesis by N-nitroso compunds 8. Conclusions - the importance of N-nitroso compunds as environmental carcinogens and as experimental models for investigating cancer Index.

The carcinogenic effects of combinations of methapyrilene hydrochloride (MP), nitrosodiethylamine (NDEA), and phenobarbital (PB) or partial hepatectomy (PH) were examined following sequential treatment of rats. MP is a generally non-genotoxic liver carcinogen of moderate potency, NDEA is a genotoxic liver carcinogen, PB is primarily a liver tumor promoter and PH induces cell proliferation. The dose of each carcinogen was chosen to be below that causing significant liver tumor incidence when given singly. There were 12 protocols involving groups of 28 female rats each. Short treatments with NDEA and MP were followed by 60 weeks of PB promotion or by partial hepatectomy. Each treatment was given separately or in double combination as controls. Several animals of each group were killed at intervals during the experiment for examination of toxic effects and the presence of altered hepatic foci. In only 3 of 12 groups was there a significant incidence of rats with liver neoplasms: the two groups given three treatments: NDEA, MP and PB (86% tumors) or NDEA, MP and PH (33%), and the group receiving NDEA and MP without promotion (46%). The results clearly indicated a co-carcinogenic effect between NDEA and MP. Continuous PB potentiated tumor development, while PH did not. There was no evidence of liver toxicity from any of the treatments, but clear cell foci observed in three groups at weeks 13 and 33 correlated with the later development of liver neoplasms.

The industrial chemical glycidol is a directly acting mutagen and a broadly acting carcinogen in rats. It was administered to Syrian golden hamsters (20 male and 20 female) by gavage of 12 mg twice a week for 60 weeks. The total dose per animal was 1.45 g or 20 mmol. Survival was not different from control hamsters treated with corn oil/ethyl acetate. Of the treated males, 9 had tumors and 13 of the treated females had tumors, some of which were adrenal cortex tumors seen in controls. More tumors were seen in the glycidol-treated hamsters than in controls, but the spleen was the only notable target organ and the number of animals with spleen hemangiosarcomas was small. Glycidol appeared to be less carcinogenic in hamsters than in rats or mice.

Female Swiss mice (Cr:NIH(S)) developed bronchiolar cell hyperplasia, dysplasia, metaplasia, and various morphologic types of bronchiolar cell tumors after topical (skin) application of N-nitroso-methyl-bis-chloroethylurea (NMBCU) or N-nitroso-tris-chloroethylurea (NTCU). These compounds are the first found to induce systemically bronchiolar cell tumors in mice in high incidence. Twice a week, with a 3-day interval, a 25-microliter drop of 0.04 mol/l (molar) NMBCU or NTCU in acetone was applied to the shaved interscapular integument for a maximum of 35 to 40 weeks. The earliest lung neoplasms were seen in mice that died after 23 weeks of treatment and affected 11 of 19 with NMBCU and 14 of 19 with NTCU treatment. Tumor growth pattern was nodular or the neoplastic tissue was frequently disseminated throughout the parenchyma, starting from multicentric peribronchiolar foci. The most common tumor types were squamous cell carcinomas and adenosquamous carcinomas, followed by adenocarcinomas with or without secretory cells, and a single ciliated-cell tumor. Histochemical and immunohistochemical studies were carried out on paraffin-embedded lungs using the avidin-biotin immunoperoxidase complex procedure and antisera against keratin, Clara cell antigen, surfactant apoprotein, neuron-specific enolase, bombesin, and chromogranin A. In several mice from both groups, hyperplasias and tumors were composed of cells expressing Clara cell antigen. No tumor cells were found expressing alveolar type II or neuroendocrine cell markers. It appeared that bronchiolar cells, in particular Clara cells, had migrated from terminal bronchioles or invaded bronchiolar walls to extend into the alveolar parenchyma. Squamous cell metaplasia with keratin expression was seen within airways or associated with glandular tumors, especially at the periphery. A unique cell type, with large eosinophilic globules and associated eosinophilic crystals, was seen lining airways or forming hyperplastic and neoplastic lesions. N-nitroso-methyl-bis-chloroethylurea- and NTCU-induced mouse bronchiolar cell alterations could be an interesting new model to study mechanisms of bronchiolar cell differentiation and tumor formation.

A number of nitrosamines that have been studied by administration to rats as solutions in drinking water have been examined by gavage administration of similar doses, for assessment of the role of pharmacokinetics in organ-specific carcinogenesis. Methylnitrosoethylamine was more effective as a liver carcinogen by gavage than in drinking water and gave rise to tumors of the lung and nasal mucosa by the former route, but not the latter. By gavage, methylnitroso-2-oxopropylamine and methylnitroso-2-hydroxypropylamine induced mainly tumors of the esophagus, as they did when given to rats in drinking water, but the potency was greater by gavage. At a higher dose rate (85 micromoles per week) methylnitrosohydroxypropylamine induced a high incidence of mesenchymal tumors of the kidney and lung tumors, in addition to esophageal tumors, but methylnitrosooxopropylamine did not. The tobacco-specific carcinogen NNK induced tumors of the liver, and to a lesser extent, of the lung and nasal mucosa when given by gavage to rats, as it did by other routes of administration. The similarly basic nitrosamine methylnitroso-N, N-dimethylaminoethylamine was equally potent, whether administered by gavage or in drinking water to rats, and gave rise only to tumors of the esophagus. The cyclic nitrosamine nitrosomorpholine was equally effective by gavage and in drinking water, but induced in rats more esophageal tumors by gavage in addition to a high incidence of liver tumors. Its 2-hydroxy derivative, a postulated metabolic intermediate of nitrosodiethanolamine, was a very much weaker carcinogen than either the latter or nitrosomorpholine, and induced low incidences of liver and lung tumors toward the end of the lifespan of the rats.

To compare the susceptibilities of the skin of different strains of mice to the carcinogenic effect of a directly acting alkylating agent, groups of 20 mice were treated twice a week with 25 μl of a solution of methylnitrosourea in methanol. The solution was 0.04M and was applied to the shaved back of female BALBc. Sencar, CD-1 and Swiss mice for 25 weeks. Four groups of 20 mice of each strain were 8 weeks old at the beginning of treatment. Another four groups were 58 weeks old when treatment began. More of the BALBc mice developed skin tumors than the other three strains, the Sencar mice somewhat less. Few CD-1 mice developed skin tumors and about one third of the Swiss mice. In all four strains, there were fewer animals with skin tumors among those begun at 58 weeks than in the young mice, but the difference was small. Survival was poor among CD-1 mice, but there was not a large difference between the strains in time of appearance of first tumor, or in average latent period of skin tumors, almost all of which were carcinomas. The Sencar mice were not out-standingly more sensitive to skin carcinogenesis by MNU, as they were to UV radiation-induced skin carcinogenesis. In a comparable study in Swiss mice neither dimethylnitrosourea nor diethylnitrosourea induced skin tumors by painting and both showed only a weak systemic carcinogenic effect in the lungs, although they are directly acting mutagens.

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 methyl-nitroso-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.

Several nitrosamines and an azoxyalkane have been administered intravesically to groups of 12 female F344 rats, twice a week for 20 or 30 weeks. Many of the nitrosamines were as efficacious in giving rise to the same tumors of internal organs as when similar doses were administered orally, showing that absorption from the bladder was as rapid as from other sites. The tumors produced included lung and kidney tumors by nitrosodimethylamine, colon and Zymbal gland tumors by azoxymethane, liver tumors by methylnitrosoethylamine (but not by nitrosodimethylamine), liver and esophagus tumors by nitrosodiethylamine, liver and lung tumors by methylnitrosamino-3-pyridylbutanone, liver tumors by nitrosomorpholine, and tumors of the esophagus by methylnitroso-n-butylamine, 2,6-dimethylnitrosomorpholine and methylnitrosamino-N,N-dimethylethylamine. Bladder tumors were induced by intravesicular administration of only low doses of nitrosobis-(2-oxopropyl)amine and to a lesser extent by methylnitroso-n-hexylamine and nitroso-(2-hydroxypropyl)(2-oxopropyl)amine, which all induced tumors systemically in addition. The bladder mucosa seemed to lack enzymes necessary to activate most nitrosamines to locally acting proximate carcinogens, but was quite transparent to the passage of carcinogenic nitrosamines present in the urine into the body to induce tumors in distant organs.

A number of asymmetric nitrosodialkylureas containing ethyl, hydroxyethyl, 2-hydroxypropyl, 2-oxopropyl or chloroethyl on one or the other side of the nitroso function were given to male and female F344 rats in drinking water. The two compounds containing a 2-oxopropyl group, ethylnitrosooxopropylurea and oxopropylnitrosochloroethylurea were also given by gavage at the same weekly doses as in drinking water. The effect was greater following gavage treatment, both in tumor incidence and in mortality rate. The most potent carcinogen was ethylnitrosooxopropylurea which induced a large variety of tumors, including lung, nervous system, colon, intestine, thyroid, skin and uterus tumors, mammary adenocarcinomas and mesotheliomas. A similar pattern of tumors was induced by ethylnitrosohydroxyethylurea and hydroxyethylnitrosoethylurea. Hydroxyethylnitrosochloroethylurea, hydroxypropylnitrosochloroethylurea and oxopropylnitrosochloroethylurea gave rise to tumors in fewer organs. Chloroethylnitrosohydroxypropylurea was very toxic to the kidneys and induced a few lung tumors. Skin tumors were commonly induced by the nitrosodialkylureas in drinking water, but not when given by gavage.

The effects of administering similar doses of some simple alkylating agents to rats by different regimens have been compared. Two of the compounds were methylating agents, nitrosodimethylamine and azoxymethane; two were ethylating agents, nitrosodiethylamine and azoxyethane; and nitrosomethylethylamine was both a methylating and an ethylating agent. The treatments gave rise to tumors in almost all treated rats. The results indicate the importance of pharmacokinetics in determining which organs are the targets of the alkylating carcinogens.

The effect of differences in level of dietary selenium on the induction of esophageal and bladder tumors in rats by two nitrosamines was investigated. Groups of 20 female F344 rats were given a synthetic diet containing less than 0.05 ppm Se to which selenium (as sodium selenite) was added at the concentration of 0.35, 0.7, 1.4 and 2.1 ppm selenium. These four groups, plus one without added Se, were treated with 20 ml per rat per day, 5 days a week, of a solution of nitrosomethylcyclohexylamine containing 5 mg/liter. A parallel five groups were treated in the same way with a solution of nitrosomethyl-3-carboxypropylamine in drinking water containing 600 mg per liter, as drinking water. Treatment lasted 28 weeks, at which time some animals had developed tumors. A group of 20 rats fed 0, 1.4 and 2.1 ppm Se was not treated with carcinogen. Rats consuming 1.4 ppm or 2.1 ppm Se gained weight more slowly than other groups. There was no significant difference in survival between the five groups treated with each carcinogen but receiving different dietary levels of selenium. Neither was there any significant difference between groups receiving each carcinogen in the incidence of tumors of the esophagus induced by nitrosomethylcyclohexylamine or of tumors of the urinary bladder induced by nitro somethylcarboxypropylamine. Control rats on the synthetic diets did not survive as well as untreated rats eating regular chow diet. In these conditions there was no effect of dietary selenium levels on the induction of tumors in female rats by the two carcinogenic nitrosamines we used.