[CANCER RESEARCH 43, 214-221, January 1983]

Carcinogenicity of Hydroxylated Alkylnitrosoureas and ofNitrosooxazolidones by Mouse Skin Painting and byGavage in Rats1

William Lijinsky2 and Melvin D. Reuber

Chemical Carcinogenesis Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701

ABSTRACT

The carcinogenic effectiveness of a number of nitrosoal-kylamides related to nitrosoethylurea and nitroso-n-propylureahas been compared by topical application to Swiss mice andby intragastric administration to F344 rats. Nitrosohydroxy-ethylurea and the related cyclic nitrosamide, nitrosooxazoli-done, were as potent as nitrosoethylurea as skin carcinogens,although the latter was a much weaker mutagen to Salmonella.When administered P.O., nitrosooxazolidone induced mainlyforestomach tumors in rats, while nitrosohydroxyethylurea wasvery broadly acting, inducing neoplasms of the lung, forestomach, glandular stomach, colon, duodenum, and bone (osteo-genic sarcomas). Nitroso-2-hydroxypropylurea, nitroso-3-hy-droxypropylurea, and nitroso-5-methyloxazolidone were allmuch more potent carcinogens on mouse skin than was nitroso-n-propylurea, nitroso-5-methyloxazolidone being some

what less effective than the nitrosoureas; the mutagenicity toSalmonella seemed not to be quantitatively related to carcino-genicity. Nitroso-5-methyloxazolidone given p.o. to rats in

duced mainly forestomach neoplasms and a few neoplasms ofthe duodenum, whereas similar treatment of rats with nitroso-2-hydroxypropylurea induced a high incidence of neoplasms

of the thymus, some of the forestomach, and few at any othersite.

INTRODUCTION

Nitrosoalkylamides comprise a class of mutagens and carcinogens which has attracted considerable interest becausethe members require no metabolic activation and are thereforedirectly acting. Most attention has been given to the N-methyland W-ethyl derivatives of nitrosourea, nitrosourethan, and

nitrosonitroguanidine. Consequently, we have considerableknowledge of the relation between methylation and ethylationof constituents of target cells (especially of DNA) and carcino-

genesis. On the other hand, we have little or no informationabout the action of donors of other alkyl groups, which mightarise from many of the more complex nitrosamines, whichcannot easily give rise to methylating or ethylating agents.

We chose, therefore, to study the carcinogenic action of anumber of hydroxylated alkylnitrosoureas and cyclic nitrosoal-

kylcarbamates (nitrosooxazolidones) which are related to them(Fig. 1). The 2 test systems chosen were mouse skin painting,which has been used for quantitative comparison of carcino-

1This work was supported by Contract N01-CO-75380 with the National

Cancer Institute, NIH, Bethesda, Md. 20205.2 To whom requests for reprints should be addressed.

Received April 7, 1982; accepted September 30, 1982.

genie effectiveness of a number of alkylnitrosoureas (4), andstomach intubation (gavage) in rats. For quantitative comparisons, as far as possible the concentrations of compounds inthe test solutions were equimolar, and the length of treatmentwas approximately the same.

MATERIALS AND METHODS

Chemicals

NJtroso-2-hydroxyethylurea. The preparation of this compound (8)

was reported by Swenson ef al. (10) to give rise to an impure product,containing the cyclic nitrosooxazolidone; we used this fact to prepareboth compounds from ethanolamine (2-hydroxyethylamine; Aldrich

Chemical Co., Milwaukee, Wis). Twelve g (0.2 mol) ethanolamine weredissolved in 10 ml water containing 10 g ice and 20 ml 10 N HCI andcooled in ice. Sixteen g (0.2 mol) of potassium cyanate were addedslowly; there was considerable effervescence and an increase in pHfrom 3 to 7.5. The solution was then boiled for 15 min; some ammoniaevolved. After cooling, 40 ml 5 N H2SO„were added, the solution wascooled in ice, and 8 g sodium nitrite were added slowly and allowed toreact for 30 min. The yellowish pink solution was extracted with 3 x75 ml ethyl acetate, the combined extracts were shaken with 10 mlwater, dried with anhydrous magnesium sulfate, and the solvent wasremoved at 40°on a rotary evaporator. To the semisolid residue were

added 100 ml of méthylènechloride, which caused the precipitation ofa white solid. This was filtered off, washed with méthylènechloride,and allowed to dry in air. The yield was 5 g, m.p. 79-80°; UV A^?"0'

(«),395 nm (90).

C3H7N3O3

Calculated: C 27.07, H 5.30, N 31.58Found: C 26.90, H 5.17, N 31.50

Nitrosooxazolidone. The méthylènechloride solution from theabove preparation was evaporated to dryness, leaving a pink solid,which was pressed dry on filter paper. The yield was 4 g, m.p. 45-46°;UV X™r"(i), 402 nm (126).

C3H4N203

Calculated: C 31.05, H 3.49, N 24.30Found: C 30.96, H 3.35, N 23.87

Contrary to a statement in Ret. 10, the compound was not particularlyunstable and remained unchanged in the freezer (—20°)for several

years and in solution in acetone or ethanol for several days.Nitroso-2-hydroxypropylurea (Nitrosoisopropanolurea). The prep

aration of this compound and the following one was similar to that fornitrosohydroxyethylurea and nitrosooxazolidone described above. Fifteen g (0.2 mol) isopropanolamine (Aldrich Chemical Co.) were dissolved in 20 ml HCI diluted with 20 ml water and cooled in ice; 16 g(0.2 mol) potassium cyanate were added slowly, after which the solution was boiled for 15 to 20 min. After acidifying with 40 ml 5 N H2SO4,the solution was cooled in ice, and 12 g sodium nitrite were added.

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Carcinogenicity of Alkylnitrosoureas and Nitrosooxazolidones

After reacting for 30 min, the solution was extracted with 3 x 75 mlethyl acetate, the extract was washed with water and dried withanhydrous magnesium sulfate, and the solvent was removed. To thesemisolid residue, 100 ml of méthylènechloride were added, the whitesolid was filtered off, washed with méthylènechloride and dried, andthe méthylènechloride solution was used for the next preparation. Theyield of white solid, nitroso-2-hydroxypropylurea, was 3 g, m.p.81-83°; UV AS"0' (e). 396 nm (93).

Calculated: C 32.65. H 6.1 6, N 28.56Found: C 32.79, H 6.28, N 28.53

Nitroso-5-methyloxazolidone. The residue, after evaporation of the

méthylènechloride solution from the previous preparation, was a pinksolid, which was pressed dry on filter paper. The yield was 5 g, m.p.36-38°; UV X^,ancl (e), 402 nm (1 28).

Calculated:Found:

C 36.92, H 4.65, N 21 .54C 37.54, H 4.90, N 20.95

Nitroso-3-hydroxypropylurea. The preparation was identical withthat for nitrosoisopropanolurea, except that 1-amino-3-propanol was

used instead of isopropanolamine. Beginning with 0.1 mol of thehydroxyamine and 0.1 mol of potassium cyanate, with the other constituents in proportion, the yield of white solid was approximately 2 g,m.p. 56-58°; UV ASE^U), 396 nm (79).

C4H9N3O3

Calculated: C 32.65, H 6.16, N 28.56Found: C 32.68, H 6.34, N 28.43

Animal Treatments

In the skin-painting experiments, each of the nitrosoalkylamides was

dissolved in acetone at a concentration of 0.04 M, and 25 nil of thesolution were applied twice each week from a calibrated pipet to theshaved interscapular region of each of a group of 20 female Swissmice. The mice were from the colony of the NCI-Frederick Cancer

Research Facility and were bred and housed 10 to a plastic cage withwire mesh bottom within a barrier facility. The mice were 6 to 7 weeksold at the beginning of treatment, which was continued for 40 to 50weeks, as specified in Table 1 . The back of each mouse was carefullyexamined once a week, and all tumors and other lesions were chartedon graph paper. At death, each animal was necropsied, and the skin ofthe application site was fixed for histological examination. There wasa concurrent group of acetone-treated control mice and a group of

untreated control mice.In addition to the skin-painting experiments, the 2 nitrosooxazoli-

dones, nitrosohydroxyethylurea and nitroso-2-hydroxypropylurea,were administered i.g.3 to groups of Fischer 344 rats, also obtained

from the colony of this facility and maintained in a barrier facility;treatment of the rats began when they were 6 to 8 weeks old. All of therats were male and were treated in groups of 20, housed 4 to a plasticcage with wire mesh bottom. The animals were given Rockland rat dietin pellets and water ad libitum. Twice a week each animal was given bygavage 0.2 ml of a solution of one of the nitrosoalkylamides dissolvedin corn oil containing 30% ethyl acetate (the 2 nitrosohydroxyureaswere not sufficiently soluble in corn oil alone). The concentrations ofthe compounds in the oil-ethyl acetate are given in Table 2. The

experiments were not all begun at the same time and, because theconcentrations of the nitrosooxazolidones were somewhat lower thanthose of the nitrosohydroxyureas, a second set of gavage experimentswith lower concentrations of the latter were begun. In Table 2 are giventhe concentrations of the compounds in the solutions and the length of

3 The abbreviation used is: i.g., intragastrically.

treatment in each case. At the end of treatment, the animals wereallowed to die naturally (a few were killed when moribund) and werethoroughly necropsied. All lesions and major organs and tissues werefixed for histological examination. Because of the report by Pelfrene etal. (8) of the induction of bone tumors by nitrosohydroxyethylurea inrats, special attention was paid to the bones of these animals andsections of the femurs were taken in all cases, but it was not possibleto examine the animals radiologically. Only tumors that rarely or neverappear in control F344 rats were considered to be a consequence ofthe treatment.

The mutagenicity tests were carried out as was described for a largegroup of nitrosoalkylureas (1) and used Salmonella typhimurium strainTA 1535. The nitrosoureas were dissolved in dimethyl sulfoxide, andthe solutions were serially diluted so that doses of 1 to 500 ¿igwerecontained in 0.1 ml, which was added to 2 ml of molten agar and 0.2ml of bacterial suspension. This solution was poured on the surface of20 ml of VBE agar, and the plates were incubated at 37° in the dark

for 48 hr, after which the revertan! colonies were counted. Each assaywas conducted at least twice, and from the average numbers ofrevertants a dose-response curve was constructed. The straight-line

ascending portion of the graph was used to calculate the number ofrevertants per ¿umolof mutagen, as given in Table 1.

RESULTS

Table 1 shows the results of the skin painting of Swiss micewith the 5 nitrosoalkylamides, together with these reportedpreviously (4) for nitrosoethylurea and nitroso-n-propylurea,which can be considered the "parents" of the oxygenated

compounds. The results of a mutagenicity test in S. typhimurium strain TA 1535 of the compounds are also given and showvery large differences in mutagenic potency between the nitrosoalkylureas and their oxygenated derivatives. Some discrepancies between results and those reported previously (1) canbe attributed to the presence of cyclic nitrosocarbamates asimpurities in the older preparations of nitrosohydroxyal-

kylureas.As in our previous skin-painting study, the neoplasms ap

pearing on the skin are given as cumulative totals of animalswith one or more skin neoplasms at 5-week intervals, until no

more appeared or all animals were dead. Several animals hadmore than one skin neoplasm, but these are not enumerated.Virtually no neoplasms regressed, and most of the mice withskin neoplasms had at least one carcinoma, as well as papillonnas in many cases. Pathological examination of each tumorwhen several were present was not always carried out. Inaddition to mice with squamous cell carcinomas, some hadtrichoepitheliomas, rhabdomyosarcomas, or neurosarcomas ofthe skin. In a few animals, skin neoplasms were not diagnosedbecause of cannibalism.

There was no great difference in the dose of each compoundreceived by the mice, so that the number of animals withneoplasms and the rate at which they appeared can be considered a reasonable index of relative carcinogenic potency. Bythis measure, nitrosohydroxyethylurea and nitrosooxazolidonewere not statistically different in potency (as analyzed by Cox's

2-tailed test) from nitrosoethylurea, although nitrosooxazoli

done was somewhat slower in inducing tumors. On the otherhand, the 3 derivatives of nitroso-n-propylurea were enor

mously more potent than was the parent compound, whichitself seemed to be a rather weak skin carcinogen in mice.There was no statistically significant difference in effectivenessbetween nitroso-2-hydroxypropylurea and nitroso-3-hydroxy-

JANUARY 1983 215

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propylurea, as measured by either rate of appearance of skinneoplasms or total number of animals with skin neoplasms.Statistical analysis showed that nitroso-5-methyloxazolidone

was less effective than the 2 nitrosohydroxypropylureas butwas more effective than nitrosooxazolidone (p = 0.023).

In Table 2 is described the study in which the nitrosoalkylam-ides were given p.o. to F344 rats, including the survival recordof each group of animals and the doses administered. All ofthe treatments led to early death of the rats with neoplasmscompared with our untreated animals, of which, characteristically, 80% would be alive at 100 weeks of such an experiment(106 weeks of age). The 2 nitrosooxazolidones seemed to givea lower mortality rate than did the nitrosohydroxyalkylureas,even though the total dose of the former was greater than thelatter, and although the concentration of the nitrosooxazolidones was only a little higher. Nitrosomethyloxazolidone led toearlier death of the rats than did nitrosooxazolidone at thesame dose rate, which can be considered an index of greaterpotency. On the other hand, there was not a greatly reducedmortality in the rats given the 2 nitrosohydroxyalkylureas at thelower dose rates, compared with the higher dose rates, whichwere 2.5 times larger, but given for 25 weeks compared with30 weeks. Nitroso-2-hydroxypropylurea caused more rapiddeath than did nitrosohydroxyethylurea at both dose rates.

Most of the rats in all treatment groups died with neoplasmsthat can be considered induced by the treatments (Table 3).These neoplasms generally resulted in death. The most common neoplasm induced by nitrosooxazolidone and by nitroso-5-methyloxazolidone was carcinoma of the forestomach (non-

glandular stomach). These carcinomas were large, and theyinvaded or metastasized to the peritoneum, liver, spleen, andlymph nodes. Both compounds induced a small number ofneoplasms of other organs, but apart from the 4 large invasivecarcinomas in the duodenum induced by the 5-methyl com

pound, the incidence of none of these could be considered tobe significant. Nitroso-2-hydroxypropylurea induced undiffer-entiated leukemia involving the thymus, which was responsiblefor the relatively rapid death of these animals. The thymicleukemias and lymphosarcomas (9) were different from themonocytic leukemia commonly found in untreated F344 rats.Nitrosohydroxypropylurea also induced some neoplasms of theforestomach, more at the higher dose rate than at the lower,and a small number of lung neoplasms. None of the otherneoplasms that appeared in animals treated with this compoundwas in significant incidence.

In marked contrast with the 2-hydroxypropyl compound,

nitrosohydroxyethylurea was an extremely broadly acting carcinogen, inducing at both dose rates a large array of neoplasmsthat do not appear in untreated rats of this strain. There weremore large carcinomas of the forestomach which invaded andmetastasized in the animals given the higher dose rate, but theincidence of the remaining neoplasms did not differ betweenthe 2 dose rates. Leukemias involving thymus were notablyabsent. The commonest neoplasms seen in the rats givennitrosohydroxyethylurea were adenocarcinomas of the lung,but there were also adenocarcinomas of the colon and duodenum, and several osteogenic sarcomas which metastasizedto the lungs. It is possible that the incidence of the latter washigher than we have reported, because the animals were notX-rayed and small tumors in the bones could have been missed.Three animals given the lower dose of nitrosohydroxyethylurea

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Carcinogenicity of Alkylnitrosoureas and Nitrosooxazolidones

Table 2

Survival of male F344 rats given nitrosohydroxyalkylureas and nitrosooxazolidones twice weekly by gavage

Total dose No. of rats at

CompoundNitroso-2-hydroxyethylureaNitrosooxazolidoneNitroso-2-hydroxypropylureaNitroso-5-methyloxazolidoneUntreated"

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had transitional cell papillomas of the bladder, in addition toother neoplasms.

The principal neoplasms were characterized as follows.Undifferentiated Lymphoma of Thymus with or without

Leukemia. The thymus was markedly enlarged. The corticalportion of the entire thymic architecture was replaced by adiffuse cell pattern made up of relatively large cells containinglarge round to oval nuclei with distinct nucleoli and scantycytoplasm (Fig. 1). Scattered among these cells were largephagocytic histiocytes with cytoplasm containing cell debris.Capsular invasion was observed.

Cells similar to those in the thymus generally were alsopresent in the spleen, liver, bone marrow, and other organs inrats with leukemia.

Adenocarcinoma of the Small Intestines. The carcinomaswere large and invaded the muscularis and serosa. They consisted of glands of varying sizes lined by columnar cells. Nucleiwere pseudostratified and cytoplasm was basophilic (Fig. 2).In some areas, the glands were separated by thin strands ofconnective tissue; in other areas, the connective tissue wasdense.

Osteogenic Sarcoma. Large osteogenic sarcomas invadedthe adjacent skeletal muscle. The sarcoma was made up ofsheets of cells which often made osteoid (Fig. 3). Métastaseswere present in other organs, including the lungs (Fig. 4).

Adenocarcinoma of the Lungs. Neoplasms of the lungsvaried from well differentiated to poorly differentiated. Thebetter-differentiated tumors were made up of glands; the less

differentiated of sheets of cells (Fig. 5). Glands often containedmucin. The carcinomas metastasized to the adrenal glands andother organs (Fig. 6).

Adenocarcinoma of the Colon. Carcinomas containedglands with column cells, pseudostratified nuclei, and basophilic cytoplasm supported by stems (Fig. 7).

Squamous Cell Carcinoma of the Forestomach. Carcinomas metastasized to the peritoneum, liver, and other organs(Fig. 8).

DISCUSSION

Skin painting of a chemical on mouse skin is one of thesimplest ways of quantifying a carcinogenic effect and has longbeen used for comparing carcinogenic potency of large numbers of polynuclear aromatic compounds, which presumablyhave to be metabolically activated to a carcinogenic form. Wehave used the same test system to compare the carcinogeniceffectiveness of a number of nitrosoalkylureas (4), which are

presumed to form alkylating agents directly without need ofmetabolic activation. There was in that study no obvious relationship between the effectiveness of the nitrosoalkylurea as acarcinogen and the ease with which it gave rise to an alkylatingagent (possibly an alkyl ion or radical), as measured by itsdirect mutagenicity to Salmonella. In the present experimentswith the oxygenated derivatives of nitrosoalkylureas, there isno quantitative relationship between mutagenicity and carcinogenic activity on mouse skin. Nitrosohydroxyethylurea isabout equally potent as a carcinogen with nitrosoethylurea,and nitrosooxazolidone appears somewhat less potent, yetboth nitrosooxazolidone and nitrosohydroxyethylurea are 2orders of magnitude more potent mutagens than is nitrosoethylurea. Nitroso-n-propylurea is a weak carcinogen for mouseskin and a relatively weak mutagen, although it is more muta-genie than nitrosoethylurea, which is a strong carcinogen.Nitroso-3-hydroxypropylurea is a weaker mutagen than is ni-troso-n-propylurea but is a much more potent carcinogen thanthe latter. The carcinogenicities in mouse skin of nitroso-3-hydroxypropylurea, nitroso-2-hydroxypropylurea, and nitroso-5-methyloxazolidone are similar, although both nitroso-2-hy-droxypropylurea and nitroso-5-methyloxazolidone are very potent mutagens in Salmonella, and the 3-hydroxypropyl com

pound is a weak mutagen. Since all of these compounds aredirectly acting, the bacterial mutagenesis results are not quantitatively informative about direct carcinogenesis by this groupof carcinogens. If initiation of neoplasms in the skin begins withformation of an alkylating agent interacting with DNA (or othermacromolecule), other factors are surely involved in development of neoplasms, since the commonly used phorbol esterpromotors are not very effective in causing such initiated cellsto develop into neoplasms of the skin" (11 ).

Considering the results of p.o. administration of the oxygenated derivatives of nitrosoethylurea and nitroso-n-propylurea

presented here, the current theories of chemical carcinogenesis are clearly incomplete in explaining them. As illustrated inChart 1, nitrosohydroxyethylurea and nitrosooxazolidone mightbe expected to give rise directly to the same alkylating moiety(2-hydroxyethyl-) as would be derived by a-oxidation of nitro-sodiethanolamine, by analogy with the early studies of Mageeand Farber and their successors of the simple dialkylnitrosa-

mines (5). However, nitrosodiethanolamine is a liver carcinogenin rats, inducing mainly hepatocellular carcinomas (3), andpresumably requires activation in the liver, whereas neithernitrosohydroxyethylurea nor nitrosooxazolidone gives rise to

4 E. Hecker and W. Lijinsky. unpublished information.

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Carcinogen/city of Alkylnitrosoureas and Nitrosooxazolidones

H2N-C-NCH2CH2OH

ON.CH2

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Chart 1. Structure of nitrosohydroxyalkylureas and nitrosooxazolidones.

any liver neoplasms in rats. This is reminiscent of the fact thatneither nitrosomethylurea nor nitrosoethylurea gives rise toliver neoplasms when given to intact adult rats i.g. or evenintraportally (2), even though they undoubtedly produce alkyl-

ated macromolecules in liver cells; these nitrosoalkylureas giverise to neoplasms at many other sites (2, 6). The questionarises why the 2 presumed precursors of hydroxyethyl- do not

induce liver neoplasms but induce them at so many other sites,when nitrosodiethanolamine does induce liver neoplasms inrats. It is obvious that the compounds, or a transport form ofthe hydroxyethyl-, reach many other organs and tissues and

cells within them, in which they induce neoplasms, presumablyby direct action. In particular, nitrosooxazolidone induces manyneoplasms of the forestomach, but few elsewhere, whereasnitrosohydroxyethylurea has an extremely broad carcinogenicaction, inducing not only neoplasms of the forestomach, butalso lung, colon and kidney tumors and osteogenic sarcomas.There is no statistically significant difference in incidence ofneoplasms of the forestomach between the groups treated withthe 2 nitrosooxazolidones and the higher concentration ofnitrosohydroxyethylurea. The incidence of these neoplasms inthe rats treated with nitrosohydroxyethylurea and nitroso-2-

hydroxypropylurea shows a highly significant trend with doseas shown by Cox's exact regression test of proportions.

Induction of osteogenic sarcomas has been reported byPelfrene ef a/. (8), although they used impure nitrosohydroxyethylurea as imputed by Swenson ef a/. (10). The latter authorsreported the induction mainly of thymic leukemias in mice bynitrosohydroxyethylurea, a type of neoplasm almost totallyabsent from our rats given this compound, although theseneoplasms were induced by the higher homologue, nitroso-2-hydroxypropylurea. It is possible that we underestimated thenumber of bone neoplasms induced in our rats, perhaps because our examination of the bones was not as thorough asthat carried out by Pelfrene era/. (8). Nevertheless, that findingof a rare neoplasm induced in rats by this particular nitrosocompound has been confirmed in our study, and it leaves thedilemma of the failure of nitrosooxazolidone, chemically sosimilar to nitrosohydroxyethylurea, to induce the same spectrum of neoplasms, so far unresolved. One explanation mightbe that nitrosohydroxyethylurea and nitrosooxazolidone do notgive rise to the same alkylating moiety. Indeed, decompositionof nitrosooxazolidones in base is very complex, giving rise to

acetylenes, aldehydes, and ketones, among other products(7).

Similarly, the difference in effect of nitroso-2-hydroxypro-pylurea and of nitroso-5-methyloxazolidone and their failure toinduce similar neoplasms in rats to those induced by nitroso-bis(2-hydroxypropyl)amine is difficult to explain. The lattercompound by «-oxidation would be expected to give rise to a2-hydroxypropylating agent, the same moiety to which theformer 2 nitrosoalkylamides would be expected to give risedirectly. However, nitrosobis(2-hydroxypropyl)amine inducesmainly esophageal neoplasms in rats, whereas the nitrosam-

ides do not, even though this organ of F344 rats is particularlysusceptible to the induction of tumors. Instead, nitroso-5-meth-

yloxazolidone induces neoplasms of the forestomach in almostall of the treated rats, together with a few in the duodenum,while nitroso-2-hydroxypropylurea induced many neoplasmsof the thymus (none of which were induced by the chemicallysimilar nitroso-5-methyloxazolidone), together with some neo

plasms of the forestomach and a few of the lung.These profound differences in carcinogenic effectiveness

between A/-nitroso compounds which might be expected to actthrough the same common active reagent suggest that such ahypothesis is inadequate as an explanation of the mechanismsof carcinogenesis by these compounds. It might be that reactions of the whole molecule of the nitrosoalkylamides areinvolved in carcinogenesis and that distribution of these molecules, as might be affected by their particular structures, is thedeterminant of their action in particular cells.

ACKNOWLEDGMENTS

We thank A. W. Andrews for the mutagenesis assays in Salmonella. C. W.Riggs for the statistical analysis of the results, and Barbara Thomas and DanLogsdon for expert technical assistance.

REFERENCES1. Lijinsky. W.. and Andrews, A. W. The mutagenicity of nitrosamides in

Salmonella typhimurium. Mutât.Res.. 68: 1-8, 1979.2. Lijinsky, W., Garcia, H., Keefer, L, and Loo, J. Carcinogenesis and alkylation

of rat liver nucleic acids by nitrosomethylurea and nitrosoethylurea administered by intraportal injection. Cancer Res., 32. 893-897, 1972.

3. Lijinsky. W.. Reuber, M. D., and Manning. W. B. Potent carcinogenicity ofnitrosodiethanolamine in rats. Nature (Lond.). 288 309-310, 1980.

4. Lijinsky, W., and Winter, C. Skin tumors induced by painting nitrosoalkylureas on mouse skin. J. Cancer Res. Clin. Oncol., 102: 13-20, 1981.

5. Magee, P. N., and Farber, E. Toxic liver injury and carcinogenesis. Methyl-ation of rat liver nucleic acids by dimethylnitrosamine in vivo. Biochem. J.,83: 114-124, 1962.

6. Magee, P. N.. Montesano, R., and Preussmann, R. N-Nitroso compoundsand related carcinogens. In: C. E. Bearle (ed.). ACS Monograph Series. No.173, pp. 491-625. Washington, D. C.: American Chemical Society. 1976.

7. Newman, M. S.. and Kutner, A. New reactions involving alkaline treatmentof 3-nitroso-2-oxazolidones. J. Am. Chem. Soc.. 73. 4199-4204. 1951.

8. Pelfrene, A.. Mirvish. S. S., and Gold. B. Induction of malignant bone tumorsin rats by 1-(2-hydroxyethyl)-1-nitrosourea. J Nati. Cancer Inst., 56.445-446, 1976.

9. Singer. S. S., Singer, G. M.. Saavedra, J. E.. Reuber, M. D., and Lijinsky.W. Carcinogenesis by derivatives of 1-nitroso-3,5-dimethylpiperazine in rats.Cancer Res., 41: 1034-1038, 1981.

10. Swenson. D. H., Frei, J. V., and Lawley. P. D. Synthesis of 1-<2-hydroxy-ethylj-t-nitrosourea and comparison of its carcinogenicity with that of 1-ethyl-1-nitrosourea. J. Nati. Cancer Inst., 63. 1469-1473, 1979.

11. Waynforth, H. B., and Magee, P. N. The effect of various doses andschedules of administration of W-methyl-N-nitrosourea, with and withoutcroton oil promotion, on skin papilloma production in BALB/c mice. GannMonogr. Cancer Res., 17: 439-488, 1975.

JANUARY 1983 219

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Fig. 1 Undifferentiated lymphoma of the thymus. Thymus is replaced by relatively large cells. Cells have large, round nuclei with distinct nucleoli and scantycytoplasm. H & E, x 680.

Fig. 2. Adenocarcinoma of the duodenum. Carcinoma is made up of glands lined by columnar cells with pseudostratified nuclei and basophilic cytoplasm. H & E,x 190.

Fig. 3. Osteogenic sarcoma of the femur. The cells grow in sheets and make osteoid. H & E, x 400.

Fig. 4. Metastatic osteogenic sarcoma to the lungs. Osteoid is present in the métastases. H & E, x 180.

220

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-7Fig. 5. Adenocarcinoma of the lungs. In some areas, the carcinoma consists of glands; in other areas, there are sheets of cells. H & E. x 180.

Fig. 6. Metastatic adenocarcinoma of the lung to adrenal. Carcinoma has replaced adrenal cortex. H & E, x 200.

Fig. 7. Adenocarcinoma of the colon. Glands contain column cells, pseudostratified nuclei, and basophilic cytoplasm. H & E, x 180.

Fig. 8. Metastatic squamous cell carcinoma of the forestomach to the liver. H & E, x 180.

221

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1983;43:214-221. Cancer Res   William Lijinsky and Melvin D. Reuber  RatsNitrosooxazolidones by Mouse Skin Painting and by Gavage in Carcinogenicity of Hydroxylated Alkylnitrosoureas and of

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